cAMP-dependent protein kinase 1

Effects of Mutation or Deletion

Clones of cells deficient in Pka-C1, were induced by mitotic recombination. Derivatives of all imaginal discs examined, including wings, halteres, legs and antenna, frequently exhibit duplications that include marked PKA mutant clones. Three features are common to all wings with PKA mutant clones extending to the wing margin. First, ectopic tissue and pattern alterations are induced only by clones in the anterior compartment and involve only anterior cells. Second, mutant clones alter the growth and fate of neighboring cells. Third, the extent of induced pattern alterations increases with the distance of the PKA mutant clone from the AP compartment boundary. These three principles also apply to phenotypes observed in legs. Transcription of decapentaplegic and other genes normally expressed at the AP compartment boundary is induced ectopically in anterior PKA mutant clones, and transcriptional induction of dpp does not require normal hh activity (Li, 1995).

Likewise Pka mutant clones in the larval eye imaginal disc, located anterior to the morphogenetic furrow, ectopically induce dpp expression, whereas clones located posterior to the furrow fail to do so. Thus PKA is required to repress dpp expression in eye disc cells anterior to the location at which dpp is normally expressed. A PKA dominant negative mutant ectopically activates dpp in the wing disc, and is a dominant suppressor of partial loss-of-function hh mutation in the eye. Pka-C1 mutant cells anterior to the morphogenetic furrow induce ectopic furrows. These furrows propagate away from the clone boundary and into the neighboring nonmutant cells (Li, 1995).

PKA mutant clones in imaginal discs express dpp and wg. Null dpp alleles suppress the majority of pattern defects in wings, notum, halteres and antennae that are observed in PKA mutants. Likewise strong mutant wingless alleles suppress PKA mutant phenotypes in ventral regions of the leg (Li, 1995).

Very little information is available about gene expression during the larval period, a developmental interval critical to the formation of the adult. To what extent does gene expression during this period resemble that in the embryonic stages, and how does gene expression during the larval period contribute to segment polarity in the adult? In fact, all the genes expressed during embryonic segment polarity also play a similar role in the formation of the adult. Cells destined to form the cuticle of the adult abdomen are present as clusters of small, non-dividing diploid cells (the anterior dorsal, posterior dorsal and ventral histoblast nests) located at stereotyped postions in the larval epidermis. These cells, just as do their embryonic counterparts, express engrailed, hedgehog, wingless, patched, cubitus interruptus and sloppy paired in a stereotyped manner dependent on their positions within each segment. Each segment is subdivided into an anterior (A) and posterior (P) compartment, distinguished by activity of the selector gene engrailed (en) in P but not A compartment cells. The ventral epidermis of each abdominal segment forms a flexible cuticle, the pleura, with a small plate of sclerotised cuticle, the sternite, centered on the ventral midline. The pleura is covered with a uniform lawn of hairs, all pointed posteriorly, whereas the sternite contains a stereotyped pattern of bristles. Posterior compartments are to a large degree devoid of hairs and bristles, while the sternite cuticle of the A compartment consists of an anterior-to posterior progression of six types of cuticle distinguished by ornamentation and pigmentation. Just anterior to the posterior compartment, A6 is unpigmented, with hairs and none of the larger ornaments called bristles. A5 is darkly pigmented with hairs and bristles of large size. A4 and A3 are darkly and lightly pigmented respectively with moderately sized hairs and bristles. A2 is lightly pigmented with hairs, and A1, adjacent to the next more anteriorly located "posterior" compartment is unpigmented without hairs (Struhl, 1997a).

A second paper (Struhl, 1997b) deals directly with the instances in which cell polarity does not correspond to the presumed concentration gradient of Hh and considers whether Hh acts directly or by a signal relay mechanism. In some cases various manipulations cause non-autonomous effects on cell polarity, a vectorial property. For example, PKA mutant clones in the A3 and A4 region alter polarity of hairs and bristles both within the clone and outside it. In general, wild-type cells positioned laterally and posteriorly to the mutant clone form hairs and bristles that point centripetally towards the clone; thus, behind the clone, cells form hairs and bristles that point anteriorly. The region of wild-type tissue showing this reversed polarity can be up to 4 cell diameters wide. Of great interest is the effect of PKA and smo mutant clones in the anterior portion of A compartment. PKA and smo mutant clones in the anterior region of the A comparment alter cell type much as they do in the posterior portion, but some clones of smo cells in the A1 region form hairs that have reversed polarity and these hairs point forward. Consequently, it is surmised that Hh influences cell polarity indirectly, possibly by inducing other signaling factors (Struhl, 1997b).

Evidence is presented that Hh does not polarize abdominal cells by utilizing either Decapentaplegic or Wingless, the two morphogens through which Hh acts during limb development. If Hh were to work through Wg to influence polarity, removal of wg from clones of cells that are activated in the Hh pathway should eliminate that influence. Neither the change in cell type nor the alterations in cell polarity cause by the loss of PKA activity appear to be due to the ectopic expression of wg. Likewise, eliminating dpp from PKA mutant clones fails to alter the polarity phenotype (Struhl, 1997b).

How might Hh polarize cells via a signal-relay mechanism? One clue is that within and surrounding some PKA mutant clones the hairs and bristles point inwards, towards the center. A simple model is that the loss of PKA activity in these cells mimics reception of Hh and hence induces them to secrete a diffusible polarising factor, 'X'. Because mutant cells in the center of the clone would be surrounded by X-secreting cells, they might be exposed to higher levels of X than mutant cells at the periphery. If cells were oriented by the direction of maximal change (the vector) in the concentration of X, cells both inside and outside of the clone would point towards the center of the clone. Such a propagation model does not demand that X be diffusible, because polarity could be organized by local cell-cell interactions, which spread as in a game of dominoes (Struhl, 1997b).

The movement of the morphogenetic furrow is dependent upon the secretion of the signaling protein Hedgehog (Hh) by more posterior cells. It has been suggested that Hh acts as an inductive signal to induce cells to enter a furrow fate and begin differentiation. Nevertheless, hh loss-of-function clones have a negligible effect on furrow progression. To further define the role of Hh in the process of furrow progression, clones of cells were examined lacking the function of the smoothened gene. smo is required for transduction of the Hh signal and allows the investigation of the autonomous requirement for hh signaling. These experiments demonstrate that the function of hh in furrow progression is indirect. Cells that cannot receive/transduce the Hh signal, by virtue of being smo mutants, are still capable of entering a furrow fate and differentiating normally. This suggests that a second signal, received from adjacent cells, is required for entering a furrow fate and differentiating normally. However, hh is required to promote furrow progression and regulate its rate of movement across the disc, since the furrow is significantly delayed in smo clones. Activation of the hh pathway anywhere anterior to the furrow (as occurs in pka-C1 mutant clones) does not immediately trigger ectopic photoreceptor differentiation. The inability of pka-C1 loss-of-function clones to induce ectopic entry to furrow fate, except when close to the endogenous furrow, is not due to insufficient activation of the hh signaling pathway in the mutant cells. That is, double mutants for smo and pka-C1 have identical fates (ectopic photoreceptor differentiation) to clones mutant for pka-C1 alone. Entry into furrow fate only occurs when pka-C1 comes to lie close to the advancing furrow. This has lead to the proposal that a "zone of competence" lies immediately anterior to the furrow. The identity of the second, furrow-inducing signal is unknown, but it is possible that it is provided by a physical relay from cell to cell (Strutt, 1997).

Biochemical measurements indicate that Pka-C1 is either the sole or the major PKA catalytic subunit gene in Drosophila. Adult females heterozygous for a strong and a weak Pka-C1 allele fail to lay eggs and show a striking and novel defect in oogenesis that includes the formation of egg chambers containing multinucleate nurse cells. Females heterozygous for two weak Pka-C1 alleles are fertile but produce offspring showing a variety of defects in embryogenesis, including preblastoderm arrest and alterations in cuticular patterning. Animals zygotically null for Pka-C1 die as morphologically normal first-instar larvae, implying that maternally encoded protein, which persists and lasts for at least 12 hr, suffices for embryogenesis. Animals hemizygous for weak Pka-C1 alleles survive for several days as larvae but grow slowly (Lane, 1993).

Genetic manipulations of cut activity results in defective packaging of germline-derived cysts into egg chambers and disintegration of the structural organization of oocyte-nurse cell complexes to generate multinucleate germline-derived cells. Although these egg chambers invariably

contain 16 germline-derived nuclei, the total number of ring canals is frequently decreased to 14 or 13. The distribution of ring canals is also abnormal in affected egg chambers. cut null alleles in combination with wimp result in egg chamber defects. wimp encodes the RNA polymerase II 140 kD subunit. Ectopic cut expression produces compound egg chamber. cut interacts genetically with the Notch gene and with the catalytic subunit of Protein kinase A gene during egg chamber morphogenesis. cut null mutations suppress loss of Notch function during oogenesis. Two different cut null mutations reduce the incidence of compound egg chambers found in mutant Notch ovaries. Since cut expression is restricted to the somatic follicle cells and cut mutant germline clones are phenotypically normal, it is proposed that the defects in the assembly of egg chambers and the changes in germline cell morphology observed in cut mutant egg chambers are the result of altered interactions between follicle cells and germline cells. It is suggested that cut participates in intercellular communications by regulating the expression of molecules that directly participate in this process (Jackson, 1997).

Flies carrying a viable heteroallelic combination of mutant alleles of the gene encoding the PKA catalytic subunit, DC0, are reduced in size (Skoulakis, 1993). To test whether the adenosine 3'-5'-monophosphate (cAMP)-PKA pathway might represent a target for Neurofibromin 1, pupae of a DC0 heteroallelic combination were tested to see whether increasing PKA activity in Nf1 mutant animals would rescue the size defect. A constitutive active murine PKA* transgene was expressed in Nf1 mutant flies. Heat shock-induced expression of this transgene results in lethality. However lower transgene expression can be achieved by growing the fly cultures at 28 degrees C. Under these conditions, statistically significant rescue of the pupal size defect is observed. Because expression of activated PKA suppresses the phenotype of NF1 null alleles , PKA appears not to function upstream of Nf1 in a simple linear pathway. Therefore, PKA must either function downstream of Nf1 or in a parallel pathway (The, 1997).

Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly - Lamina precursor cells harboring mutations for either pka or ptc undergo differentiation cell-autonomously and independently of retinal innervation

The arrival of retinal axons in the Drosophila brain triggers the assembly of glial and neuronal precursors into a neurocrystalline array of lamina synaptic cartridges. Retinal axons arriving from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina target field. In the eye disc, photoreceptor cells assemble into ommatidial clusters behind the morphogenetic furrow (mf) as it moves to the anterior. The ommatidial clusters project their axon fascicles into the crescent-shaped lamina. Neuronal precursor cells of the lamina (LPCs) are incorporated into the axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow. Glia precursor cells (GPCs) are generated in two domains that lie at the dorsal and ventral anterior margins of the prospective lamina. These glial precursors migrate into the lamina along an axis perpendicular to that of LPC entry. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody staining. Like the eye, lamina differentiation occurs in a temporal progression on the anterioposterior axis. Axon fascicles from new ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow) and associate with neuronal and glia precursors in a vertical lamina column assembly. At the anterior of the lamina, at the trough of the lamina furrow, LPCs await a retinal axon-mediated signal in G1-phase and enter their terminal S-phase at the posterior margin of the furrow. Postmitotic (Dac-positive) LPCs assemble into columns at the posterior margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers as they become specified as the lamina neurons L1-L5. Lamina neurons L1-L4 form a stack in a superficial layer, while L5 neurons reside in a medial layer near the R1-R6 axon termini. These neurons arise at cell-type specific positions along the column's vertical axis. Lamina glial cells take up cell-type positions in the precartridge assemblies. Epithelial (E-glia) and marginal (Ma-glia) glia are located above and below the R1-R6 termini, respectively. Satellite glia are interspersed among the neurons of the L1-L4 layer. The Ma-glia and E-glia layers, both located ventral to the neuronal precursor column, sandwich the R1-R6 axon termini. The medulla neuropil serves as the target for R7/8 axons and is separated from the lamina by the medulla glia, situated just below the Ma-glia (Z. Huang, 1998 and references).

Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations (Z. Huang, 1998).

The Hh receptor Ptc, a multiple-pass membrane protein, and the cAMP-dependent protein kinase (PKA) normally maintain the Hh signal transduction pathway in a repressed state. Loss-of-function mutations in either of these genes mimic Hh signal reception and result in the cell autonomous activation of Hh target genes in many tissues. LPCs harboring mutations for either pka or ptc undergo differentiation cell-autonomously and independently of retinal innervation. Mutant cells anterior to the furrow do not differentiate precociously. This observation is consistent with the consequences of ectopic Hh expression in an the lamina in mutants lacking retinal innervation of the lamina. Hh expression in regions anterior to the lamina furrow does not induce precocious lamina differentiation, as though competence to respond to Hh is acquired by G1-phase LPCs at the anterior margin of the lamina furrow. Within the lamina target field, wild-type cells neighboring the pka or ptc mutant cells are never observed to express Dac. Thus activation of the Hh pathway by loss-of-function in either gene results in a strictly autonomous induction of LPC maturation. These results permit the conclusion that the terminal cell division and differentiation of LPCs both require the direct reception of the Hh signal (Z. Huang, 1998).

PKA and oogenesis: Regulation of cell proliferation and patterning in Drosophila oogenesis by Hedgehog signaling

The localized expression of Hedgehog (Hh) at the extreme anterior of Drosophila ovarioles suggests that it might provide an asymmetric cue that patterns developing egg chambers along the anteroposterior axis. Ectopic or excessive Hh signaling disrupts egg chamber patterning dramatically through primary effects at two developmental stages. (1) Excess Hh signaling in somatic stem cells stimulates somatic cell over-proliferation. This likely disrupts the earliest interactions between somatic and germline cells and may account for the frequent mis-positioning of oocytes within egg chambers. (2) The initiation of the developmental programs of follicle cell lineages appears to be delayed by ectopic Hh signaling. This may account for the formation of ectopic polar cells, the extended proliferation of follicle cells and the defective differentiation of posterior follicle cells, which, in turn, disrupts polarity within the oocyte. Somatic cells in the ovary cannot proliferate normally in the absence of Hh or Smoothened activity. Loss of protein kinase A activity restores the proliferation of somatic cells in the absence of Hh activity and allows the formation of normally patterned ovarioles. Hence, localized Hh is not essential to direct egg chamber patterning (Zhang, 2000).

Hh signaling in Drosophila generally regulates the abundance and activity of Ci proteins without altering CI mRNA levels. By contrast, vertebrate Hh homologs frequently regulate transcription of the Ci-related GLI family of transcriptional effectors. The induction of CI RNA in ptc mutant follicle cells provides the first evidence that this circuitry can also be found in Drosophila. Other consequences of altering the activity of Hh signaling components in ovarian somatic cells substantiate the hypothesis that Hh signaling activates at least two distinct intracellular pathways. One pathway, involving protection of Ci-155 from proteolysis and perhaps also release from cytoplasmic anchoring, is phenocopied by PKA and cos2 mutations. In the ovary, cos2 mutations elicit stronger phenotypes than PKA mutations, perhaps because cos2 mutations preferentially disrupt cytoplasmic anchoring of Ci-155. The second pathway increases the specific activity of Ci-155 in opposition to the inhibitory effects of Su(fu). This pathway is elicited by ptc, but not by PKA mutations and requires Fu kinase activity. In accordance with this model, PKA Su(fu) double mutant cells produce phenotypes almost as strong as for ptc mutants in ovaries, whereas ptc fu double mutant cells exhibit minimal phenotypes and PKA mutant phenotypes are not greatly altered by additional loss of Fu kinase activity. In imaginal discs high level Hh signaling to nearby cells is phenocopied by ptc mutations and requires Fu kinase activity, whereas only low level Hh signaling to more distant cells can be phenocopied by PKA mutations and does not require Fu kinase activity. PKA mutations in somatic ovarian cells can effectively substitute for Hh activity: Fu kinase activity is not essential for somatic cell proliferation and ptc mutations engender excessive Hh signaling phenotypes even in the absence of Hh activity. Hence, it is surmised that ovarian somatic cells normally undergo only low levels of Hh signaling, in keeping with the observation that the source of Hh in the germarium is separated from its target cells by several cell diameters (Zhang, 2000).

The subcellular localization and activity of Drosophila Cubitus interruptus are regulated at multiple levels: Role of PKA in Ci proteolysis

Cubitus interruptus (Ci), a Drosophila transcription factor, mediates Hedgehog (Hh) signaling during the patterning of embryonic epidermis and larval imaginal discs. In the absence of Hh signal, Ci is cleaved to generate a truncated nuclear form capable of transcriptional repression. Hh signaling stabilizes and activates the full-length Ci protein leading to strong activation of downstream target genes, including patched and decapentaplegic. A number of molecules have been implicated in the regulation of Ci. Mutations in these molecules effect changes in Ci protein level, and also influence the extent of Ci proteolysis and the expression of Ci target genes. This paper examines the regulation of Ci subcellular localization and activity. A bipartite nuclear localization signal (NLS) within Ci has been characterized. It is proposed that the subcellular distribution of Ci is affected by two opposing forces, the action of the NLS and that of at least two regions targeting Ci to the cytoplasm. The data also show that loss of PKA or Costal-2 activity does not fully mimic Hh signaling, demonstrating that Ci proteolysis and Ci activation are two distinct events which are regulated through different paths. It is proposed that there are three levels of apparent Ci activity, corresponding to three zones along the AP axis with different sets of gene expression and different levels of Hh signaling (Q. Wang, 1999).

Previous studies have demonstrated that protein kinase A plays an important role in Ci proteolysis. In PKA loss-of-function clones, Ci protein level is greatly elevated. An extract from discs carrying large numbers of such clones exhibits reduces proteolysis. Furthermore, Ci mutated at putative PKA sites is resistant to cleavage. The resolution of Ci into phosphorylation isoforms enables a direct test of the action of PKA upon Ci. When cl-8 cells are treated for an hour with H-89, a potent PKA inhibitor, a slight increase in the amount of full-length Ci is observed, accompanied by a slight decrease of Ci-75. This suggests that the basal activity of PKA is low in cl-8 cells. When cl-8 cells were treated with the PKA stimulator forskolin, within an hour a decrease of Ci-155 is observed, but there is no marked change in the IEF pattern of Ci. However, there is a dramatic shift in the western pattern when cells are simultaneously treated with forskolin and MG132. The amount of the unphosphorylated isoform remained constant, while the highly phosphorylated isoforms accumulate (Q. Wang, 1999).

Cells in the anterior compartment of wing pouch express different sets of ci target genes depending on their distance from the AP boundary. Based on the expression profile of Ci and its target genes, cells along the AP axis can be divided into three zones. The Anterior Zone consists of cells more than 8-9 cell diameters away from the boundary. Cells in this zone express low level Ci, low level Ptc and no Dpp. The Intermediate Zone, marked by expression of high level Ci, low level Ptc and high level Dpp, corresponds to cells between 8-9 and 2-3 cell diameters away from the boundary. Cells immediately adjacent to the boundary (within 2-3 cell diameters) fall into the Boundary Zone, marked by medium level Ci, high level Ptc, and medium level Dpp. The Ci regulated enhancer element/reporter 4bslacZ is also expressed in this zone. Expression of ci target genes is a consequence of the overall activity of many individual Ci peptides, which is determined by both the potency of each peptide and the number of peptides present. In the following discussion, the overall Ci activity observed for a cell, judged by the expression of target genes, is termed the 'apparent activity' of Ci. The specific activity, or potency, of each peptide is defined as its 'activation state'. In a wild-type wing disc, there are three levels of apparent Ci activity corresponding to the three zones. In the Boundary Zone, Ci has the highest apparent activity, activating both ptc and 4bslacZ to high levels. dpp expression in this region is likely subjected to a partial repression by anterior En. In the Intermediate Zone, Ci exhibits an intermediate level of apparent activity, inducing strong expression of dpp but not high level ptc nor 4bslacZ. In the Anterior Zone, Ci has the lowest apparent activity (Q. Wang, 1999).

Although there are three levels of apparent Ci activity, evidence has been found for only two activation states. 4bslacZ, whose sensitivity allows the monitoring Hh-induced Ci activation, is expressed only in the Boundary Zone, suggesting that Ci is activated only in this zone. Ci in the activated state, therefore, is given the name Ciboundary. The high level of Ci in the Intermediate Zone indicates that cells in this zone must receive some level of Hh signaling, which inhibits Ci proteolysis. Despite the high protein level, Ci in the Intermediate Zone is not sufficiently activated to induce 4bslacZ expression, and is given the name Cidefault. The lack of proteolysis in the Intermediate Zone allows Cidefault to accumulate, resulting in high level expression of dpp. In the Anterior Zone, the majority of Ci is proteolytically cleaved into Ci-75. Although the Anterior Zone and the Intermediate Zone differ in Ci protein levels and the expression patterns of target genes, at present there is no evidence that they differ in the state of Ci activation. In fact, when Ci stabilization is mimicked in the Anterior Zone by making PKA loss-of-function clones, the expression of high level dpp is also mimicked. This observation is consistent with the idea that the two zones share the same activation state of Ci (Cidefault) but differ in the levels of full-length Ci. In summary, the three levels of apparent Ci activity correspond to Ci boundary, high level Cidefault, and low level Cidefault, respectively (Q. Wang, 1999).

The boundary between the Anterior Zone and the Intermediate Zone corresponds to a division between cells with low Ci levels and those with high Ci levels, and is likely to coincide with the anterior border of Hh signaling. Between this line and the AP boundary, cells in both the Intermediate Zone and the Boundary Zone receive some level of Hh signaling and show stabilization of Ci. (The relatively lower Ci level in the Boundary Zone probably reflects partial transcriptional repression by anterior En. However, the activation from Cidefault to Cibounday happens only in the Boundary Zone, suggesting that it takes place when the level of Hh signaling is above a certain threshold. The level of Hh signaling changes across the anterior compartment. In the Boundary Zone, cells express high level Ptc, which both transduces and sequesters Hh signaling. The presence of high level Ptc creates a steep decline of the Hh signal. Consequently, cells receiving Hh are divided into those receiving high level Hh signal (the Boundary Zone) and those receiving lower level Hh signal (the Intermediate Zone). Its role in regulating Hh distribution makes Ptc essential for proper regulation of the apparent activity of Ci (Q. Wang, 1999).

Study of Hh-induced Ci activation has been complicated by the fact that in almost all the assays, a high level of Ci protein seems to suffice for the activation of Ci target genes. This is well illustrated in the case of loss-of-function PKA clones, in which elevated Ci protein levels are associated with strong activation of Hh target genes such as ptc and dpp. It has been difficult to tell whether the activation of downstream genes is due to elevated Ci alone, or if Ci is activated in addition to being stabilized in these clones. Progress was made in a recently published study through a combination of manipulating the expression of an uncleavable deletion construct of Ci and examining smo loss-of-function clones in the posterior compartment (Methot, 1999). Importantly, it demonstrates that inhibition of Ci proteolysis is not sufficient to activate Ci. However, the assays as described do not address the question of whether in vivo stabilization and activation of Ci are regulated simultaneously as two consequences of the same process, or regulated separately through different mechanisms. The sensitivity of 4bslacZ allows this question to be addressed. While a modest level of Ci in cells receiving high level Hh signal can activate 4bslacZ and clones lacking ptc activate 4bslacZ, high level Ci in PKA or cos2 loss-of-function clones cannot. It is concluded that Ci is stabilized but not activated in PKA or cos2 mutant clones. In other words, these mutations mimic one aspect of Hh signaling but not the other, and Ci in the clones exists as Ci default instead of Ci boundary (Q. Wang, 1999).

A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast: PKA and CAP functionally cooperate in the germline to control actin organization

A mutation in a novel gene, capulet (cap), was identified in a mosaic screen to isolate mutations that perturb actin organization in germline clones. Adenylate cyclase-associated proteins (CAPs) have been shown to inhibit actin polymerization in vitro, by sequestering monomeric actin. This actin-binding activity has been mapped to the carboxy-terminal region of CAP; however, a 'verprolin homology'-related domain has been identified in all CAPs, just carboxy-terminal of the polyproline-rich domain. In members of the verprolin/WASP family, this motif binds actin monomers in vitro, but catalyses actin polymerization in vivo. Therefore, in CAP homologues, this region of the protein may be used to facilitate actin binding. As CAP proteins have also been found associated with Abl tyrosine kinase and with adenylate cyclase, it is possible that CAP represents an intermediary in these signal transduction cascades, perhaps altering actin dynamics in response to extracellular cues (Baum, 2000).

The genetic screen also identified a mutation in the catalytic subunit of protein kinase A (PKA). Therefore, pka and cap mutant phenotypes in the Drosophila germline were compared. Like the cap mutant, pka germline clones lose nurse cell cortical actin, while simultaneously accumulating ectopic actin structures. In addition, the pka mutant phenotype is sensitive to the dosage of CAP, and actin defects are dramatically enhanced in pka;cap double germline clones. These data suggest that PKA and CAP functionally cooperate in the germline to control actin organization (Baum, 2000).

In cap germline clones, F-actin accumulates in a highly polarized fashion within the egg chamber and oocyte. Thus, whether loss of CAP perturbs other aspects of normal polarity, including the asymmetric localization of mRNAs within the oocyte was investigated. The distributions of bicoid and oskar mRNAs, which localize to anterior and posterior poles of the oocyte, respectively, were examined. Although oskar mRNA is concentrated in one region of the oocyte in over 90% of egg chambers, oskar mRNA is mislocalized in 76% of stage 8-10 cap germline clone egg chambers. Moreover, in 28% of cases, oskar transcripts are localized to the anterior or lateral part of the oocyte. In addition, in 64% of stage-10 egg chambers that maintain correct overall polarity, oskar mRNA has a diffuse distribution and is not tightly focused at the posterior pole. The localization of bicoid transcripts was also examined. bicoid mRNA accumulates at an aberrant site in 65% of cap mutant egg chambers, and is localized to the posterior pole in 36% of stage 8-10 egg chambers. Thus, cap germline clones display two related mRNA polarity defects: (1) although oocytes are able to concentrate oskar and bicoid mRNAs locally within the oocyte, they appear unable to coordinate mRNA polarity with the morphological polarity of the egg chamber; (2) in the majority of egg chambers in which oskar mRNA is correctly transported to the posterior pole of the oocyte, oskar message is not tightly localized at the cortex (Baum, 2000).

It can be concluded that CAP is a major regulator of actin dynamics in Drosophila, and that CAP is likely to function to inhibit actin polymerization in vivo, as it does in vitro. A striking feature of the cap phenotype is the accumulation of actin filaments at polar sites within the egg chamber. This cannot be explained by differences in the monomeric actin pool in nurse cells versus the oocyte, as G-actin, as measured by DNaseI staining, is equally distributed within the egg chamber, as is profilin. Thus, CAP inhibits actin filament formation at specific cellular sites, possibly in response to signaling events (Baum, 2000).

In both yeast and multicellular eukaryotes, the actin cytoskeleton responds to cell signaling events. Therefore it is interesting to note that homologs of Drosophila CAP have been shown to interact physically with an Abl tyrosine kinase and adenylate cyclase. These latter proteins transduce extracellular cues, in a way that is not fully understood, to remodel the actin cytoskeleton within the growth cones of migrating neurons to facilitate axon guidance. Thus, CAP may constitute part of the machinery that reorganizes the actin cytoskeleton in response to these signals in neurons and in other polarized cells. Interestingly, the genetic screen also identified the catalytic subunit of protein kinase A (PKA), which acts downstream of adenylate cyclase, as a gene required for proper actin organization and oocyte polarity. Since yeast, Hydra and human CAPs have been shown to facilitate the activation of adenylate cyclase, CAP and PKA may be elements of a conserved signal transduction pathway. The phenotypic similarities shared by cap and pka germline clones suggest that CAP and PKA act together in the Drosophila female germline. Given this interaction, CAP could be a substrate for PKA, or could facilitate the activation of adenylate cyclase upstream of PKA. Alternatively, because a reduction in both CAP and PKA activity leads to a more severe phenotype, the two genes may act in parallel pathways. CAP and PKA are, however, unlikely to be essential components in a common signal transduction pathway in Drosophila because no evidence is found for related CAP and PKA functions in somatic tissues (Baum, 2000).

In existing mutants known to perturb the germline actin cytoskeleton, oocyte polarity is either unaffected or completely disrupted. Therefore, whether oocyte polarity is altered in the cap mutant was investigated by examining the localization of both bicoid and oskar mRNAs. When compared to other known mutants, cap germline clones exhibit novel mRNA polarity defects (although similar defects are exhibited by pka null germline clones). First, cap mutant oocytes are able to localize mRNAs to discrete areas within the oocyte, but the sites of mRNA deposition do not respect the existing morphological axes of the egg chamber. Second, in the majority of stage-10 egg chambers with the correct polarity, oskar mRNA is observed in a shallow gradient, as if diffusing away from the cortex at the posterior pole. Thus, CAP seems to be required, both to coordinate mRNA localization with the axial polarity of the egg chamber, and to tether mRNAs to the cortex. Because microtubules are thought to mediate the transport of mRNAs to opposite poles of the oocyte in the wild type, the defect in oocyte axial polarity in the cap mutant may result from defects in the underlying microtubule cytoskeleton. cap germline clones frequently contain a misoriented microtubule array, with plus ends focused at the anterior cortex. This altered microtubule polarity is therefore probably responsible for the mislocalization of oskar and bicoid mRNAs at early stages of oogenesis. At later stages, following disassembly of the polar microtubule array, an actin-based structure at the posterior pole of the Drosophila oocyte, dependent on CAP and tropomyosin, may act as a tether to hold oskar mRNA at the cortex (Baum, 2000).

Interactions between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting in Drosophila: Feedback inhibition through protein kinase A on the InsP3 receptor by increased levels of 20-hydroxyecdysone

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. Using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting have been examined in Drosophila. Previous studies have shown that mutants for the Inositol 1,4,5-trisphosphate receptor gene (Itpr) are larval lethals. In addition, they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Mutants for adenylate cyclase (rut) synergize, during larval molting, with Itpr mutant alleles, indicating that both cAMP and InsP3 signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP3 receptor. Furthermore, these studies predict the existence of feedback inhibition through protein kinase A on the InsP3 receptor by increased levels of 20-hydroxyecdysone (Venkatesh, 2001).

Since ecdysone secretion occurs as tightly regulated peaks, preceding each molt, inherent in the system should be a mechanism that inhibits ecdysone secretion once the peak level has been reached. On the basis of data from the UAS-mC* transgene (coding for a dominant active form of PKA), it is suggested that increased levels of 20-hydroxyecdysone in the hemolymph initiate a negative feedback loop that requires PKA activation and inhibition of the InsP3 receptor. Thus the activated PKA phenotype is not rescued by increased levels of 20-hydroxyecdysone, but is rescued by increased levels of the itpr transgene. Interestingly, the effect of the UAS-mC* transgene on molting is also lost when Itpr gene levels are reduced as in larvae of the genotype UAS-mC*/+; 1664GAL4/itpr90B0. This observation supports the idea that the Itpr gene is downstream of the UAS-mC* effect, and in addition suggests that the negative feedback is highly sensitive to levels of the Itpr gene. While these results demonstrate interactions between the two signaling pathways, the molecular basis of these interactions is unknown as yet. Since mammalian InsP3 receptors can be directly phosphorylated by PKA, the possibility exists that a similar mechanism might operate in the negative feedback step predicted from these results. However, both predicted isoforms of the Drosophila InsP3 receptor, which are present in larval tissues and derive from two known splice variant forms of the itpr cDNA, lack putative PKA phosphorylation sites as determined by Prosite analysis. It is possible that a low-abundance isoform of the InsP3 receptor exists in specific larval cells that may be directly regulated by PKA. Additionally, there are almost certainly other unidentified players in this system that this study has not revealed. It should be possible to identify some or all of these factors using suitable genetic interaction screens in the future (Venkatesh, 2001).

Signaling upstream of PKA: Genetic analysis of the Drosophila Gsalpha gene

One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gsalpha, the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. The identification of 'loss-of-function' mutations in the Drosophila Gsalpha gene (dgs) is reported in this study. Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gsalpha protein. Examination of mutant alleles representing loss-of-Gsalpha function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells (Wolfgang, 2001).

Embryos deficient in PKA show a variety of morphological defects, including alterations in cuticular patterning. To compare the effect of individual dgs mutations on embryonic pattern formation, cuticles were examined from late stage mutant embryos or early first instar larvae generated from germline clones, as well as those zygotically mutant. All dgs mutants that hatched had normal cuticle patterns. In addition, most dead embryos also had normal cuticle patterns, with the exception of dgsB19 mutant embryos generated from germline clones. In this case, of the mutant embryos that did not hatch, ~30% showed defects in the telson formation, 30% exhibited telson defects combined with posterior abdominal segment defects, and 30% were wild type. However, since only 4% of the total dgsB19/dgsR19 mutant population fail to hatch, the actual percentage of dgsB19 mutant embryos and first instar larvae showing patterning defects is only ~3%, similar to that observed for other alleles (1%-4% of the total mutant population). Thus, ~95%-99% of all mutant embryos show normal cuticular patterning (Wolfgang, 2001).

Observation of dgsB19 mutant larvae on egg plates and in bottles indicates that they have sluggish, uncoordinated movements. To quantify larval mobility and activity, the behavior of homozygous dgsB19 third instar larvae was assayed using the 'rover' assay. Individual larvae were placed on an egg plate uniformly coated with yeast and, after 5 min, the length of the track left in the yeast by the larvae was measured. These data were then represented as histograms showing the number of larvae (y-axis) that crawled a given distance (x-axis). Although there is some individual overlap, as a population, larvae homozygous or hemizygous for dgsB19 crawl shorter distances than heterozygous controls. There appears to be no dominant effect of the mutation on activity since dgsB19/CyO-GFP individuals show crawling activity similar to a variety of controls. The reduced crawling observed for dgsB19/dgsB19 homozygotes was rescued by introduction of the dgs transgene. During this analysis, it was noted that some dgsB19 mutant larvae often crawl in continuous circles, backward, or lie on their backs for extended periods of time, behaviors that are not observed in heterozygous or nonmutant larvae. It was also noted that dgsB19 mutants appear to not be attracted to yeast granules on the egg plates, suggesting sensory-motor deficits. These results indicate that dgsB19 mutant larvae have severe defects in neural and/or muscle physiology (Wolfgang, 2001).

The phenotypes generated by mutations in the dgs gene in Drosophila stand in direct contrast to those generated by mutations in the DC0 gene, encoding a presumed downstream effector of this pathway, PKA. For example, females carrying germline clones for DC0 mutations fail to lay eggs. In various heteroallelic combinations, PKA mutant females show defects in oogenesis due to the disruption of microtubule distribution and the localization of RNAs encoding key determinants (e.g., bicoid and oskar) along the anteroposterior axis. In contrast, adult females whose germlines carry dgs null alleles (e.g., dgsR60 and dgsR19) lay morphologically normal eggs that develop to late embryonic stages with ~50% hatching. Furthermore, embryos deficient in PKA also show a variety of morphological defects including preblastoderm arrest and alterations in cuticular patterning. Patterning defects arise due to the role of PKA as a modulator of the conversion of Cubitus interruptus (Ci), the transcription factor responsible for transducing signals mediated by the morphogen Hedgehog, from a transcriptional activator to a transcriptional repressor; in PKA-deficient embryos, Ci is not processed to the repressor form, resulting in phenotypes resembling ectopic hedgehog expression. In contrast, embryos maternally or zygotically deficient for Gsalpha do not show patterning defects consistent with alterations in the hedgehog signaling pathway. In addition, direct measurement of cAMP levels in larvae homozygous for dgs null mutations (dgsR60 and dgsR79) has shown that signaling through Gsalpha plays a major role in establishing basal levels of this second messenger. Since dgs mutations do not generate the embryonic patterning defects observed in DC0 mutants, basal PKA activity is likely not to depend on pathways activated by Gsalpha that contribute to basal levels of cAMP. Interestingly, alterations observed on a physiological level following mutational and pharmacological manipulation of cAMP levels cannot be mimicked, or are not observed to the same extent, following partial, mutational inactivation of PKA. These observations suggest that PKA activation by cAMP in Drosophila, at least in these developmental contexts, does not proceed by receptor-mediated modulation of the activity of adenylyl cyclase and increased intracellular cAMP. This interpretation is also consistent with two other observations: (1) phenotypes generated by expression of constitutively active forms of Gsalpha could not be suppressed by genetic and biochemical elimination of PKA activity; (2) expression of a cAMP-independent form of PKA in both embryos and imaginal discs is able to rescue phenotypes generated by elimination of PKA activity. These results are consistent with the conclusion that the activity of PKA, again at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells. Since PKA is activated by cAMP, these observations leave open the question of how activation of PKA is mediated or of the role of cAMP in these contexts. However, these observations do not address the role of PKA in the generation of phenotypes present in dgs mutants (Wolfgang, 2001).

The results presented here further support the notion that Gsalpha-mediated activation of adenylyl cyclase in the production of cAMP and the activation of PKA are not necessarily coupled in a linear or dependent fashion. Three general alternatives can be invoked as the underlying basis for the differential response to the elimination of Gsalpha vs. PKA. (1) These genetic studies may point to the existence of a novel cAMP-independent signal transduction pathway activated directly by Gsalpha. Indeed, activation of cAMP-independent pathways by Gsalpha has been proposed in a variety of mammalian cell systems. (2) Alternatively, since expression of a constitutively activated form of Gsalpha in cultured mammalian cells results in increased intracellular cAMP, it may be that the primary mediator of the effects of cAMP in these cellular contexts is a molecule other than PKA, such as cyclic nucleotide-gated channels. In addition, (3) since gamma-subunits are potent modulators of a number of biochemical processes, it is possible that free ßgamma, generated as a consequence of the absence of Gsalpha, may in fact be responsible for some phenotypes associated with dgs mutations. Clearly, a goal of future studies will be to differentiate between these formal alternatives (Wolfgang, 2001).

Drosophila Costal1 mutations are alleles of protein kinase A that modulate hedgehog signaling

Hedgehog (Hh) signaling is crucial for the development of many tissues, and altered Hh signal transduction can result in cancer. The Drosophila Costal1 (Cos1) and costal2 (cos2) genes have been implicated in Hh signaling. cos2 encodes a kinesin-related molecule, one component of a cytoplasmic complex of Hh signal transducers. Mutations in Cos1 enhance loss-of-function cos2 mutations, but the molecular nature of Cos1 has been unknown. Previously identified alleles of Cos1 actually map to two separate loci. Four alleles of Cos1 appear to be dominant-negative mutations of a catalytic subunit of protein kinase A (pka-C1) and the fifth allele, Cos1A1, is a gain-of-function allele of the PKA regulatory subunit pka-RII. PKA-RII protein levels are higher in Cos1A1 mutants than in wild type. Overexpression of wild-type pka-RII phenocopies Cos1 mutants. PKA activity is aberrant in Cos1A1 mutants. PKA-RII is uniformly overproduced in the wing imaginal disc in Cos1A1 mutants, but only certain cells respond by activating the transcription factor Ci and Hh target gene transcription. This work shows that overexpression of a wild-type regulatory subunit of PKA is sufficient to activate Hh target gene transcription (Collier, 2004).

Two different map locations have been published for Cos1. Neither polytene band 30C nor 46E is consistent with the 50A1-50A2 map location for Cos1. Cos1 was placed at 50A1-50A2 due to the discovery of a deficiency that deleted the region and resulted in wing duplications when in trans to a recessive allele of cos1. It seems quite possible that a third gene was mapped that has genetic and phenotypic characteristics similar to the two Cos1 genes that have been mapped. It was assumed in those studies that the Cos1 phenotype results from haplo-insufficiency and that the Cos1 phenotype is due to the deficiency and not to a second site mutation. For example, along with the deficiency, the chromosome may carry an allele of cos2 that caused the observed phenotype (Collier, 2004).

pka-C1 is identified as a candidate for the gene mutated in Cos12, Cos13, Cos18, and Cos19. Sequence changes at the DNA level are detected at the pka-C1 locus, and these changes translate to substitutions in the coding sequence. These point mutations could influence PKA activity in several possible ways. (1) They could render PKA-C1 catalytically inactive and give dominant phenotypes due to haplo-insufficiency. (2) Alternatively, the point mutations could destabilize the encoded protein to such a degree that the mutant protein would be degraded and thus function as a protein null. This scenario would also result in dominant phenotypes due to haplo-insufficiency. These haplo-insufficiency explanations are unlikely to be correct. pka-C1 is a recessive negative regulator of Hh signaling and heterozygosity for pka-C1 null alleles or for deficiencies that delete pka-C1 does not result in any obvious phenotype. In addition, the mutations could render PKA-C1 constitutively active. The unregulated activity could be responsible for the dominant wing duplication phenotypes. However, expression in the wing imaginal disc of a mutant PKA-C1 that cannot be regulated by cAMP interferes with the normal expression pattern of the Hh target gene ptc, indicating that constitutive PKA-C1 activity antagonizes Hh signal transduction (Collier, 2004).

Therefore the possibility is favored that pka-C1Cos1-2, pka-C1Cos1-3, pka-C1Cos1-8, and pka-C1Cos1-9 encode dominant-negative (dn) versions of PKA-C1 that produce stable, full-length protein with reduced catalytic activity. There are precedents for the formation of dn pka-C1 mutants in Drosophila. A previously described dominant mutation that causes wing duplications maps near pka-C1. Although the molecular nature of this mutation was not ascertained, it was assumed that the mutation was a pka-C1 allele. Overproduction of a catalytically inactive mutant form of PKA-C1 in an otherwise wild-type background results in wing duplications, indicative of inappropriate activation of Hh signaling (Collier, 2004).

PKA-C1, like all other protein kinases, contains a catalytic core. Within the catalytic core of PKA-C1 and all kinases are defined subdomains that contain conserved amino acids and that play known roles in catalyzing the phosphate transfer reaction. The amino acids affected in pka-C1Cos1-2, pka-C1Cos1-3, pka-C1Cos1-8, and pka-C1Cos1-9 mutants are part of the catalytic core and are likely to be required for PKA-C1 catalytic activity (Collier, 2004).

pka-C1Cos1-8 contains a substitution of D for G at aa 189 (all numbering of amino acids is taken from the protein sequence of Drosophila PKA-C1). This amino acid is the third amino acid in the DFG triad located in subdomain VII. This triad is conserved in essentially all serine/threonine and tyrosine kinases. In PKA, the triad has been implicated in binding the metal ion that coordinates the ß- and gamma-phosphoryl oxygens of the ATP used as a phosphate donor. Mutation of the D residue in the triad renders PKA inactive. Therefore, it is likely that the substitution in pka-C1Cos1-8 results in a catalytically inactive kinase (Collier, 2004).

pka-C1Cos1-3 contains a K substitution for E173. This amino acid lies in subdomain VI between D169 and N174, two residues implicated in ATP binding. This region is implicated in substrate binding specificity as most serine/threonine kinases share the consensus 169-DLKPEN-174. Conversely, the sequence in most tyrosine kinases is DL(R/A)A(A/R)N. The substitution in pka-C1Cos1-3 may render PKA-C1 unable to recognize and phosphorylate serine and threonine residues. Inability to recognize appropriate substrates would render PKA-C1 catalytically inactive (Collier, 2004).

pka-C1Cos1-2 and pka-C1Cos1-9 contain a substitution of K for E at aa 130 in the sequence 128-GGEMF-132. These amino acids are near the start of subdomain V, a subdomain that is not well conserved at the sequence level among protein kinases. However, this region may be structurally important. Crystal structures of kinase domains reveal two large lobes: an N-terminal lobe and a C-terminal lobe. The N-terminal lobe consists mainly of an antiparallel ß-sheet and an alpha-helix. The C-terminal lobe is mainly helical but in PKA does contain four short ß-strands. These two lobes are separated by a linker and form a pocket in which the substrate is phosphorylated. E130 is the first residue in the first alpha-helix in the C-terminal lobe. A substitution of a basic K for the acidic E could disrupt the secondary structure, change the orientation of the N and C lobes relative to each other, and thus influence the structure of the catalytic cleft (Collier, 2004).

The ability of a catalytically impaired kinase to function in a dn fashion can be explained in several ways. For example, a catalytically inactive kinase could still bind target proteins and thus actually compete with the functional kinases for substrate. An informative example of dn proteins comes from studies of growth factor receptors. Many growth factor receptors possess kinase activity and function as dimers. Production of a catalytically inactive subunit essentially 'poisons' each dimer that it forms, leading to a dn phenotype. The inactive PKA holoenzyme consists of a tetramer of two R and two C subunits, but cAMP binding results in release of active C subunits that function as monomers. Therefore, catalytically active PKA-C1 is not part of a dimer and is not likely to be inactivated by virtue of association with a damaged subunit. However, the damaged subunit might interfere with release of an active catalytic subunit after cAMP binding. It is also possible that released catalytic PKA-C1, although not a dimer, might function as part of a larger multiprotein complex. In this case, a catalytically inactive PKA-C1 could still incorporate into complexes and render the entire complex ineffective (Collier, 2004).

pka-RIICos1A1 was recovered in a screen using gamma-rays as a mutagen. Unlike most chemical mutagens, which tend to cause point mutations, gamma-rays tend to produce larger aberrations such as chromosome deficiencies or rearrangements. A change consistent with a 100-kb inversion was found in the 5' UTR of pka-RII in pka-RIICos1A1 mutants. No other changes in RII coding sequence were detected but higher levels of RII were found in pka-RIICos1A1 mutant cells. On the basis of cos2 clone results, this is not due to regulation of pka-RII by Hh signaling. In addition, pka-RIICos1A1 clones located in the posterior compartment of the wing disc have high levels of RII but do not express Ci, the only transcription factor known to function in Hh signaling. Taken together, the data indicate that the high levels of RII observed in the wing disc are unique to pka-RIICos1A1 mutants. pka-RII overexpression is likely to cause the inappropriate activation of Hh target gene transcription observed in pka-RIICos1A1 mutants. Previous work has shown that overexpression of a mutant RI that cannot bind cAMP can activate Hh target gene transcription. The data presented here are the first to show that the other regulatory subunit found in Drosophila, pka-RII, can influence Hh signaling and that overproduction of a wild-type R subunit of PKA is sufficient to activate Hh target gene transcription (Collier, 2004).

Antibody staining for RII in wing imaginal discs facilitated the observation that RII levels are increased in pka-RIICos1A1 mutants but did not allow examination of absolute levels or possible post-translational modifications. Therefore Western analysis was used to examine RII protein levels in pka-RIICos1A1 mutant embryos. RII protein exists in two forms due to differential phosphorylation of a consensus PKA phosphorylation site in RII. In Drosophila whole-fly extract, RII can be labeled by radiophosphate at low, but not high, levels of cAMP. Increasing the cAMP concentration leads to the dissociation of R and C subunits, so the phosphorylation probably occurs when the RII subunit is part of a heterotetrameric holoenzym. In pka-RIICos1A1 mutants, the majority of the extra RII is the faster-migrating, nonphosphorylated form. This suggests that in pka-RIICos1A1 mutants, RII is in great excess relative to C subunits and RII is not phosphorylated because it is not in holoenzymes. In this situation, essentially all PKA-C1 is predicted to be in a complex with RII (Collier, 2004).

Unlike the four alleles pka-C1Cos1-2, pka-C1Cos1-3, pka-C1Cos1-8, and pka-C1Cos1-9 that are likely to be antimorphic and function as pka-C1-dominant negatives, pka-RIICos1A1 is likely to be a hypermorphic gain-of-function allele. Genetically, increased pka-RII function is predicted to mimic loss of pka-C1 function. The wing duplications caused by Gal4-driven overexpression of pka-RII are, exactly as predicted, similar to the effects of pka-RIICos1A1. Overproduction of RII could influence PKA-C1 activity in two ways: (1) RII could inhibit the catalytic activity of PKA by binding to the catalytic subunit; (2) R subunits can associate with A-kinase anchoring proteins (AKAPs) that localize PKA holoenzymes to specific subcellular locations. RII overproduction could mislocalize PKA-C1 within the cell via association with various AKAPs (Collier, 2004).

The most likely way for overproduced RII to influence Hh signaling is by reducing PKA catalytic activity. The results of PKA kinase assays are consistent with this hypothesis. In the heterotetrameric PKA holoenzyme, R subunits have two roles: inhibiting the catalytic activity of C subunits and protecting C subunits from degradation. Loss of R subunits results in increased basal activity of PKA due to the presence of free catalytically active C subunits, but in a decrease in cAMP-induced PKA activity because the C subunits are not protected from degradation. In pka-RIIEP(2)2162 mutants, RII levels are reduced by 95% and cAMP-induced PKA catalytic activity is reduced by almost 60%, indicating that C subunits have been degraded. In pka-RIICos1A1 mutants, high levels of RII should protect C subunits from degradation. At basal levels of cAMP, the stabilized pool of C subunits should remain associated with RIIs and the overall effect would be a decrease in basal PKA activity compared to wild type, which is precisely what was observed (Collier, 2004).

With the addition of cAMP, RII subunits should release C subunits and PKA activity should increase. At high levels of cAMP, essentially all C subunits should be released from RII-mediated inhibition. In this situation, mutants should contain higher levels of PKA activity than wild type because the high levels of RII have protected C subunits from degradation, resulting in an overall increase in the amount of C subunits. For the most part, measurements of PKA activity in pka-RIICos1A1 compared to wild type were exactly as predicted: basal PKA activity was decreased in pka-RIICos1A1 mutants compared to wild type while cAMP-induced PKA activity was vastly higher in pka-RIICos1A1 mutants than in wild type. However, at the lowest level of cAMP tested, a slight reduction in PKA activity from the basal level was observed for pka-RIICos1A1 mutants. This contrasts to the wild-type situation, where any added cAMP results in PKA activity higher than basal levels. One possible explanation for the slight decrease of PKA activity in the mutant could be that the initial release of a small amount of C subunits triggers the degradation reaction. At higher levels of cAMP, this effect is masked because more C subunits are released than can be immediately processed by the degradation machinery. Although RII overexpression results in higher levels of cAMP-induced PKA activity compared to wild type, the basal level of PKA activity is lower in the mutant than in wild type (Collier, 2004).

In the future, it will be interesting to examine the roles of wild-type R subunits in Hh signal transduction. If pka-RII normally plays a role in transducing the Hh signal, pka-RII mutants should possess Hh phenotypes. The only reported pka-RII mutant, pka-RIIEP(2)2162, reduces adult RII levels by greater than 95% and is homozygous viable with no phenotypes indicative of aberrant Hh signaling. RI and RII may function redundantly. Another possibility is that residual levels of RII may be sufficient for normal regulation of Hh target gene transcription. The roles of R subunits in transducing the Hh signal may be revealed only by the creation of the double mutant carrying null alleles of RI and RII (Collier, 2004).

The wing duplication phenotype of pka-RIICos1A1 indicates that Hh target gene transcription has been inappropriately activated in response to pka-RII overexpression in the anterior compartment of the wing imaginal disc. Although RII protein levels are uniform throughout pka-RIICos1A1 heterozygous wing discs, Ci is stabilized and dpp is transcribed only in the presumptive duplication. Not every cell in the presumptive duplication that contains high levels of full-length Ci transcribes dpp. The high levels of full-length Ci should indicate that Ci repressor levels are low, so dpp, at the very least, should be derepressed. This sensitivity of only certain cells to high levels of full-length Ci resulting from RII overexpression was also observed when very high levels of RII were generated in pka-RIICos1A1 homozygous clones or by GAL4-UAS-mediated expression. In general, anterior-most cells are the most sensitive to pka-RII overexpression. One possible explanation is that more medial cells contain higher levels of cAMP and thus higher levels of RII are required to suppress PKA-C1 activity (Collier, 2004).

A similar situation is observed in clones mutant for a component of the SCF ubiquitin ligase complex, Roc1a. In Roc1a mutant clones, dpp is transcribed only in the most anterior clones even though high levels of full-length Ci are detected in all anterior clones. This is surprising because the appearance of full-length Ci in these cells must correspond to a decrease in Ci75 repressor levels. In the absence of Ci75, dpp should be transcribed because it is derepressed. One possible interpretation is that in medial cells less full-length Ci is stabilized in response to pka-RII overexpression or loss of Roc1a. Because full-length Ci forms at the expense of Ci75, small reductions in full-length Ci levels translate into small increases in Ci75 levels. This small increase in Ci75 could provide enough repressor activity to repress dpp. Immunofluorescense may not be sensitive enough to detect subtle differences in full-length Ci levels between medial and lateral cells (Collier, 2004).

These studies of Costal1 provide a powerful genetic link between the kinesin-related molecule Cos2 and PKA. The genetic interaction may be due to the sensitive balance between Ci, which is modified by PKA, and Cos2, or to additional activities that more directly link PKA to Cos2. In imaginal disc development precisely controlled Hh signal transducer activities are crucial for pattern formation, building the perfect forms of wings and legs (Collier, 2004).

Effects of mutation: PKA and behavior

Neural circadian pacemakers can be reset by light. The resetting mechanism may involve cyclic nucleotide second messengers. Pacemaker resetting and free-running activity rhythms have been examined in Drosophila dunce and Pka-C1 mutants. dnc mutants exhibit augmented light-induced phase delays and shortened circadian periods, indicating altered pacemaker function. However, light-induced phase advances are normal in dnc, suggesting a selective effect on one component of the pacemaker resetting response. Also, there are circadian rhythms in cAMP content in head tissues; dnc mutations increase the amplitude of daily cAMP peaks. Thus cAMP levels are not chronically elevated in the dnc mutant. A role for cAMP signaling in circadian processes is also suggested by an analysis of Pka-C1 mutants, which have severe kinase deficits and display arrhythmic locomotor activity (Levine, 1994).

Drosophila mutants for the DCO gene, which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), display arrhythmic locomotor activity, strongly suggesting a role for PKA in the circadian timing system. This arrhythmicity might result from a requirement for PKA activity in photic resetting pathways, the timekeeping mechanism itself, or downstream effector pathways controlling overt behavioral rhythms. To address these possibilities, the protein and mRNA products from the period gene were examined in PKA-deficient flies. PER protein and mRNA undergo daily cycles in the heads and bodies of DCO mutants, indistinguishable from those observed in wild-type controls. These results indicate that PKA deficiencies affect the proper functioning of elements downstream of the Drosophila timekeeping mechanism. The requirement for PKA in the manifestation of rhythmic activity was preferentially greater in the absence of environmental cycles. However, PKA does not appear to play a universal role in output functions because the clock-controlled eclosion rhythm is normal in DCO mutants. These results suggest that PKA plays a critical role in the flow of temporal information from circadian pacemaker cells to selective behaviors (Majercak, 1997).

Upon exposure to ethanol, adult Drosophila display behaviors that are similar to acute ethanol intoxication in rodents and humans. Within minutes of exposure to ethanol vapor, flies first become hyperactive and disoriented and then uncoordinated and sedated. After approximately 20 min of exposure they become immobile, but nevertheless recover 5-10 min after ethanol is withdrawn. cheapdate, a mutant with enhanced sensitivity to ethanol, has been identified as a contributory factor, using an inebriometer to measure ethanol-induced loss of postural control. An inebriometer is a device that allows a quantitative assessment of ethanol-induced loss of postural control. The inebriometer is an approximately 4 ft long glass column containing multiple oblique mesh baffles through which ethanol vapor is circulated. To begin a "run," about 100 flies are introduced into the top of the inebriometer. With time, flies lose their ability to stand on the baffles and gradually tumble downward. As they fall out of the bottom of the inebriometer, a fraction collector is used to gather them at 3 min intervals, at which point they are counted. The elution profile of wild-type control flies follows a normal distribution; the mean elution time (MET), approximately 20 min at a standard ethanol vapor concentration, is inversely proportional to their sensitivity to ethanol. A genetic screen was carried out to isolate P element-induced mutants with altered sensitivity to ethanol intoxication using the inebriometer as the behavioral assay. One X-linked mutation isolated in this screen was named cheapdate (chpd) to reflect the increased ethanol sensitivity displayed by hemizygous mutant male flies. chpd males elute from the inebriometer with a MET of 15 min compared with 20 min for the wild-type controls. This increased ethanol sensitivity of chpd males was observed at all ethanol vapor concentrations tested. Genetic and molecular analyses reveals that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, it is found that the enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol (Moore, 1998).

Flies carrying mutations in three molecules involved in cAMP signaling were tested for response to ethanol: (1) rutabaga (rut), encoding the Ca2+-calmodulin-sensitive AC; (2) dunce (dnc), encoding the major cAMP-phosphodiesterase (PDE), and (3) DCO, encoding the major catalytic subunit of cAMP-dependent protein kinase (PKA-C1). Males hemizygous for rut mutations display an ethanol-sensitive phenotype similar to that of amn mutants. Flies heterozygous for the loss-of-function DCO alleles, which show reduced cAMP-stimulated PKA activity, also display increased ethanol sensitivity (homozygotes cannot be tested because they die as embryos). Ethanol sensitivity of males hemizygous for dnc mutations, however, are nearly normal. These data show that flies unable to increase cAMP levels normally (such as rut and possibly amn) or to respond properly to increased cAMP levels (such as DCO/+) are more sensitive to ethanol-induced loss of postural control. The converse, however, is not observed; dnc flies, whose cAMP levels are several times higher than wild type, display nearly normal ethanol sensitivity, a phenotype that is also observed in males doubly mutant for dnc and amn. Unexpectedly, whereas both rut and amn are ethanol sensitive, males doubly mutant for rut and amn are not significantly different from control (Moore, 1998).

To further investigate the relationship between cAMP signaling and ethanol sensitivity, the adenylyl cyclase (AC) activator forskolin was used to manipulate cAMP levels in adult flies. Control and amnchpd males were fed a 10 µM forskolin solution for 2 or 4 hr prior to assaying their ethanol sensitivity in the inebriometer. Whereas forskolin treatment has no effect on the behavior of control flies, the ethanol sensitivity defect of amnchpd flies is reversed by a 2 hr forskolin treatment. Likewise, treatment of rut1 males with forskolin for 2 hr leads to normal ethanol sensitivity, a result likely due to the activation of another AC. Interestingly, a 4 hr forskolin treatment of amnchpd males further reduces ethanol sensitivity, suggesting that one or more components of the cAMP pathway may have undergone compensatory up-regulation in amnchpd mutants, thereby increasing the system's ability to respond to pharmacologically induced increases in cAMP levels. Taken together, these data indicate that the effects of amn and rut on ethanol sensitivity are directly related to their ability to modulate cAMP levels (Moore, 1998).

A reduction of PKA-C1 function, as observed in males heterozygous for DCO alleles, leads to increased ethanol sensitivity. To corroborate a role for PKA in ethanol sensitivity, adult control and amnchpd males were fed solutions containing 200 µM Rp-cAMPS or Sp-cAMPS for 2 hr prior to their assay in the inebriometer. Rp-cAMPS is a competitive antagonist of cAMP that binds the regulatory subunit of PKA without releasing the catalytic subunit; Sp-cAMPS is an analog of cAMP that activates PKA. Sp-cAMPS treatment of control males does not alter ethanol sensitivity. This treatment, however, completely reverses the enhanced ethanol sensitivity of amnchpd. In contrast, feeding Rp-cAMPS to control males results in increased ethanol sensitivity. Rp-cAMPS treatment has the opposite effect on amnchpd males, partially reversing their increased ethanol sensitivity. While unexpected, this last observation is consistent with the finding that flies doubly mutant for rut and amn do not (unlike single mutants) display increased ethanol sensitivity. Treatment of control flies with the PKA inhibitor Rp-cAMPS for only 2 hr leads to an ethanol-sensitive phenotype similar to that of amn, rut, and DCO/+ flies. This argues that even a relatively short-term inhibition of the cAMP pathway is sufficient to increase ethanol sensitivity (Moore, 1998).

In mammalian cells and tissues, ethanol potentiates receptor-mediated cAMP signal transduction; the mechanisms underlying this effect, however, remain poorly understood. While a direct link between cAMP signaling and ethanol-induced behaviors has not been established in mammals, the responses to acute ethanol are thought to be mediated by alterations in the function of various ligand-gated ion channels. Certain subtypes of GABAA and NMDA receptors are potentiated and inhibited by ethanol, respectively, and both these channels can be phosphorylated by PKA in cells, tissues, or heterologous expression systems. It is tempting to speculate that PKA phosphorylation of neurotransmitter receptors is altered by ethanol and that this contributes to the behavior of the inebriated animal (Moore, 1998 and references).

In Drosophila, rest shares features with mammalian sleep, including prolonged immobility, decreased sensory responsiveness and a homeostatic rebound after deprivation. To understand the molecular regulation of sleep-like rest, the involvement of a candidate gene, cAMP response-element binding protein (CREB), was investigated. The duration of rest is inversely related to cAMP signaling and CREB activity. Acutely blocking CREB activity in transgenic flies does not affect the clock, but increases rest rebound. CREB mutants also have a prolonged and increased homeostatic rebound. In wild types, in vivo CREB activity increases after rest deprivation and remains elevated for a 72-hour recovery period. These data indicate that cAMP signaling has a non-circadian role in waking and rest homeostasis in Drosophila (Hendricks, 2001).

The daily rest of flies carrying mutations and/or transgenes that alter cAMP signaling was examined at several points in the pathway. dunce flies have a mutation in the phosphodiesterase enzyme and therefore have increased cAMP. The null mutant (dncML) rests significantly less than the background yw strain. Similarly, increasing PKA activity in flies with a heat-shock-inducible transgene of the catalytic subunit of PKA significantly decreases daily rest durations compared to pre-heat-shock rest levels. Decreased adenylyl cyclase enzyme activity and thus decreased cAMP characterize rutabaga (rut) mutants, which rest more than the Canton S background strain. Similarly, S162 flies that carry a mutation that abolishes dCREB2 activity rest more than their comparison group (siblings without the mutation). The mutation is a stop codon just upstream of the basic leucine-zipper motif of the dCREB2 gene (Hendricks, 2001).

Effects of mutation: PKA and synaptic function

It is now well established that Drosophila neurons share many molecular components of the transmitter release machinery with vertebrate neurons. The release of neurotransmitter is a multistep process that involves actions of proteins associated with the synaptic vesicle and the plasma membrane, as well as cytoplasmic proteins. Some of these proteins, e.g., Synapsin, alphaSNAP, and Ca2+ channels are known to be the downstream targets of PKA. Phosphorylation of these proteins may be important for the regulation of vesicle mobilization, docking, and fusion (Yao, 2000).

In Drosophila dnc mutants, increased cAMP levels caused by the disruption of a phosphodiesterase lead to abnormalities in channel function and nerve excitability, synaptic transmission and plasticity, growth cone motility, and nerve arborization. Using the present heterologous detection system, the altered transmitter release process could be examined in developing growth cones of dnc central neurons in isolation from the influence of postsynaptic targets. Examination of PKA-RI neurons suggests that the dnc defects in ACh secretion might be mediated by PKA. These results establish a role for the cAMP cascade in the regulation of the secretion process in developing neurons before synaptogenesis. In light of the profound alterations in synaptic efficacy and activity-dependent modulation observed in mature synapses of dnc mutants, the cAMP pathway may be involved throughout the maturation process of the synapse (Yao, 2000).

The effects of decreased cAMP levels on synaptic transmission have also been extensively studied in Drosophila. Intracellular recordings at the peripheral larval neuromuscular junction have revealed that chronically lowering cAMP causes reduced neurotransmitter release, likely because of reduction of innervation rather than impairment of transmitter release. These results do not contradict results obtained from developing central neurons. It will be important to determine how reduction in cAMP concentration affects neurotransmitter releases in the Drosophila central neurons in future studies (Yao, 2000).

The prolonged ACh currents of dnc and PKA-RI neurons may be attributable to increased ACh diffusion distance and altered presynaptic release mechanisms, as discussed above. A reduced efficiency in the formation of the exocytotic fusion pore and/or a disrupted fusion machinery may account for the prolonged release events for synaptic vesicles containing similar amounts of ACh. Exocytotic efficiency may be regulated by PKA-dependent phosphorylation of vesicular, cytoplasmic, and plasma membrane proteins involved in exocytosis. Additional mutational analysis will be required to identify the specific proteins that are targeted by PKA in this process (Yao, 2000).

Although the spontaneous release in neurons of all genotypes examined does not require Ca2+ influx, the activity-dependent increase in release frequency in dnc neurons after repetitive nerve stimulation appears to depend on the external Ca2+. It has been proposed that nerve activity regulates cAMP levels, possibly mediated by intracellular accumulation of Ca2+ through repetitive nerve spikes, which can trigger the Ca2+/CaM activation of adenylyl cyclase. The activity-dependent modification of transmission at mature synapses is altered in dnc mutants. These results suggest that the cAMP pathway may mediate such activity-dependent regulation in developing neurons before synaptogenesis as well, lending support to the notion that the cAMP pathway is important in a wide variety of neuronal processes throughout development (Yao, 2000).

A novel bioassay system is described that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball is manipulated into contact with cultured Drosophila 'giant' neurons, spontaneous synaptic current-like events are registered. These events are observed within seconds after contact and are blocked by curare and alpha-bungarotoxin, but not by TTX and Cd2+, suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurs not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displays a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses (a synapse between an axon collateral of a neuron and one of the same neuron's dendrites) formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI that disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. These mutations affect the ACh current kinetics, but not the quantal ACh packet, and the release frequency is greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis (Yao, 2000).

cAMP is thought to be involved in learning process and known to enhance transmitter release in various systems. In two Drosophila memory mutants, dunce and rutabaga, the cAMP cascade is defective, and no post-tetanic facilitation is observed at the neuromuscular junction. Thus, changes in synaptic efficacy were suggested for the molecular mechanism of memory. In Aplysia, cAMP mediates changes in synaptic transmission during dishabituation, sensitization, and classical conditioning. cAMP blocks various types of K+ channels, which in turn leads to membrane depolarization and/or a prolongation of presynaptic action potentials, and finally results in an activation of voltage-gated Ca2+ channels. The long-term potentiation (LTP) at the bullfrog sympathetic ganglion and at the rat hippocampal CA3 is also mediated by cAMP and requires Ca2+ influx at presynaptic terminals. In other cases, cAMP directly enhances Ca2+ influx through modulation of Ca2+ channels. The Ca2+-independent effect of cAMP has also been demonstrated in the crayfish neuromuscular junction and in cultured mammalian CNS neurons. Thus, the effects of cAMP on synaptic transmission are diverse. Multiple mechanisms might be operating in parallel in one synapse (Yoshihara, 2000).

Using Drosophila genetics it is possible to separate the multiple mechanisms involved in the effects of cAMP on synaptic transmission. Synaptic transmission has been examined in Drosophila embryos lacking a synaptic vesicle protein, neuronal-synaptobrevin (n-syb), which is a v-soluble NSF attachment protein receptor (SNARE) protein, and required for nerve-evoked transmitter release. Even though evoked release is absent, miniature synaptic currents (mSCs) are readily observed in n-syb null mutants. Their frequency increases in response to an increase of Ca2+ concentrations in high K+ saline. A Ca2+ ionophore, A23187, also increases the mSC frequency in the presence of external Ca2+. These findings indicate that the n-syb null mutants are still capable of responding to elevations of internal Ca2+. Furthermore, in wild-type embryos cAMP increases the frequency of mSCs in the absence of external Ca2+, but does not in the n-syb null mutants. Thus, requirements for two modes of vesicle fusion, spontaneous transmitter release and nerve-evoked release, seem to be different (Yoshihara, 2000).

The preceding results, showing that in the absence of external Ca2+ cAMP enhances spontaneous transmitter release, suggest two basic features regarding the effects of cAMP on spontaneous transmitter release. (1) This pathway involves n-syb, a protein that is essential for evoked release (n-syb-dependent pathway). (2) cAMP enhancement of release is not dependent on external Ca2+. It has been asked, using the n-syb null mutant, whether cAMP also enhances spontaneous transmitter release through an increase of intracellular Ca2+ when Ca2+ is available. Further is has been asked whether PKA encoded by DC0 is involved in this enhancing effect of cAMP on transmitter release (Yoshihara, 2000).

cAMP has been shown to enhance spontaneous transmitter release in the absence of extracellular Ca2+ and n-syb is required in this enhancement (n-syb-dependent). The cAMP-induced enhancement of transmitter release was examined in the presence of external Ca2+. The intracellular concentration of cAMP was raised by application of either forskolin, an activator of adenylyl cyclase, or by 4-chlorophenylthio-(CPT)-cAMP, a membrane-permeable analog of cAMP, in the presence of external Ca2+, while recording miniature synaptic currents (mSCs) at the neuromuscular junction in n-syb null mutant embryos. The frequency of mSCs increases in response to elevation of cAMP, and this effect of cAMP is completely blocked by Co2+ (n-syb-independent pathway). In contrast, in wild-type embryos the cAMP-induced mSC frequency increase is partially blocked by Co2+. In DC0, a mutant defective in protein kinase A (PKA), nerve-evoked synaptic currents are indistinguishable from the control, but mSCs are less frequent. In this mutant the enhancement by cAMP of both nerve-evoked and spontaneous transmitter release is completely absent, even in the presence of external Ca2+. Taken together, these results suggest that cAMP enhances spontaneous transmitter release by increasing Ca2+ influx (n-syb-independent) as well as by modulating the release mechanism without Ca2+ influx (n-syb-dependent) in wild-type embryos, and these two effects are mediated by PKA encoded by the DC0 gene (Yoshihara, 2000).

In a DC0 mutant, the amplitude and Ca2+ dependency of nerve-evoked synaptic currents are not significantly different from those in wild-type, whereas the mSC frequency is lower. Conversely, in a n-syb null mutant, n-syb DeltaF33B, no nerve-evoked synaptic currents are detected, whereas mSCs are readily observable. Thus, it appears that these two modes of vesicle fusion, nerve-evoked and spontaneous, seem to have distinct requirements (Yoshihara, 2000).

Under various conditions there is a good correlation between the frequency of mSCs and the number of quanta released by nerve stimulation. In rat cerebellar synapses, there is a clear correlation between the frequency of mSCs and the amplitude of evoked synaptic currents in preparations treated with various concentrations of forskolin. In accordance with this, in wild-type embryos, forskolin increases the frequency of mSCs and the quantal content in a similar time course. Furthermore, in the DC0 mutant, the effect of forskolin is observed neither in spontaneous transmitter release nor in nerve-evoked release. These results suggest that these two modes of transmitter release are similarly affected by cAMP-PKA (Yoshihara, 2000).

cAMP is shown at the Drosophila neuromuscular junction in third instar larvae to increase the size of exo-endo cycling pool (readily releasable pool) of synaptic vesicles. The size of this pool is closely correlated with the quantal content of synaptic potentials evoked by nerve stimulation at a low frequency. Forskolin increases the mSC frequency in wild-type embryos and in newly hatched wild-type larvae. Thus, it is likely that this pool supplies vesicles for both modes (nerve-evoked and spontaneous) of transmitter release. However, the two modes dichotomize after this step. For nerve-evoked release n-syb protein is required, whereas for spontaneous fusion this protein is not of absolute necessity, although its presence facilitates spontaneous transmitter release. The cAMP-PKA cascade seems to influence the vesicle fusion process at multiple levels: (1) vesicle mobilization and translocation, which increase the size of exo-endo cycling pool; (2) modification of Ca2+ influx through voltage-gated Ca2+ channels (n-syb-independent pathway), and (3) modulation of transmitter vesicle fusion (n-syb-dependent pathway). It is likely that the first mechanism affects both modes of vesicle fusion similarly. However, the second and third mechanisms may act differentially on the two modes of vesicle fusion, which may explain the phenotype of the DC0 mutant, namely at the resting state the cAMP-PKA cascade might not be affecting the nerve-evoked transmitter release, whereas spontaneous release might be supported by the baseline activity level of the cascade (Yoshihara, 2000).

Effects of mutation: PKA and learning

The requirement for cAMP-dependent protein kinase (PKA) in associative learning of Drosophila was assessed in mutant flies hemizygous for a cold-sensitive allele, X4, of the DC0 gene, which encodes the major catalytic subunit of PKA. DC0X4 hemizygotes die as third-instar larvae at 18 degrees C, the restrictive temperature, but are viable when raised at 25 degrees C. Shifting adult DC0X4 hemizygotes from 25 degrees C to 18 degrees C leads to a decrease in PKA activity from 24% to 16% of wild-type without impairing viability. At 25 degrees C, DC0X4 hemizygotes exhibit reduced initial learning relative to controls but normal memory decay in a Pavlovian olfactory learning assay. Shifting the temperature from 25 degrees C to 18 degrees C prior to training reduces initial learning to a similar extent in DC0X4 hemizygotes and controls but results in a steeper memory decay curve only in DC0X4 hemizygotes. These observations are suggestive of a role for PKA in medium-term memory formation in addition to its previously established role in initial learning (Li, 1996).

The site-selected P-element mutagenesis of a Drosophila gene encoding the regulatory subunit of cAMP-dependent protein kinase generates mutants that have defective behavior in the olfactory learning test. The effects of these mutations in a courtship conditioning assay are described. Wild-type males can distinguish between virgin females (which they court vigorously), and fertilized females (which they court less vigorously). After exposure to fertilized females, wild-type males modify their behavior by decreasing courtship to subsequent target virgins, an effect that may last for many hours. Like wild-type males, PKA-RI mutant males are also able to distinguish between virgin and fertilized females. PKA-RI males also modify their behavior towards virgin females after prior exposure to a fertilized female, but such an effect is short-lived, suggesting a defect in memory rather than learning. Under these conditions the behavior of PKA-RI males is similar to that of amnesiac, dunce and rutabaga males (O'Dell, 1999).

The tumor-suppressor gene Neurofibromatosis 1 (Nf1) encodes a Ras-specific GTPase activating protein (Ras-GAP). In addition to being involved in tumor formation, NF1 has been reported to cause learning defects in humans and Nf1 knockout mice. However, it remains to be determined whether the observed learning defect is secondary to abnormal development. The Drosophila NF1 protein is highly conserved, showing 60% identity of its 2,803 amino acids with human NF1. Previous studies have suggested that Drosophila NF1 acts not only as a Ras-GAP but also as a possible regulator of the cAMP pathway that involves the rutabaga (rut)-encoded adenylyl cyclase. Because rut was isolated as a learning and short-term memory mutant, the hypothesis has been pursued that NF1 may affect learning through its control of the Rut-adenylyl cyclase/cAMP pathway. NF1 has been shown to affect learning and short-term memory independent of its developmental effects. G-protein-activated adenylyl cyclase activity consists of NF1-independent and NF1-dependent components, and the mechanism of the NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory (Guo, 2000).

This idea is supported by studies of NF1 mutant flies carrying a transgene encoding a mutant catalytic subunit of cAMP-dependent protein kinase (PKA*), which is constitutively active. Sustained expression of this PKA subunit rescues the small body size phenotype of NF1 mutants. Heat-shock induction of the constitutively active PKA should, in principle, bypass the requirement for the Rut-AC and all other molecules upstream of normal PKA activation. The hsp70-PKA* transgene completely rescues the learning defect of NF1P1 when the flies are raised at room temperature. NF1P2 mutants are partially rescued by the transgene at room temperature, but show complete rescue with heat shock (37°C, 30 min), or with a shift to 25°C overnight before being tested. In addition, NF1 mutations also cause a short-term memory defect (3- and 8-h retention) that is also fully rescued by heat-shock induction of PKA*. To determine whether expression of hsp70-PKA* induces a nonspecific enhancement of learning, leaky or induced expression of hsp-PKA* in the wild-type background does not increase the learning score even if flies are undertrained. For undertraining, flies were subjected to 3 repeats of electric shock in a single training trial instead of 12 trials. It is concluded that the PKA* effect is not nonspecific and that the learning defect observed in NF1 mutants can be rescued by induction of PKA activity. Therefore, the biochemical deficiency in the NF1 mutants must reside upstream of PKA induction in the cAMP pathway (Guo, 2000).

Hormones, receptors and cAMP/PKA signaling during wing expansion

At the last step of metamorphosis in Drosophila, the wing epidermal cells are removed by programmed cell death during the wing spreading behavior after eclosion. The cell death is accompanied by DNA fragmentation demonstrated by the TUNEL assay. Transmission electron microscopy reveals that this cell death exhibits extensive vacuoles, indicative of autophagy. Ectopic expression of an anti-apoptotic gene, p35, inhibits the cell death, indicating the involvement of caspases. Neck ligation and hemolymph injection experiments demonstrate that the cell death is triggered by a hormonal factor secreted just after eclosion. The timing of the hormonal release implies that the hormone to trigger the death might be the insect tanning hormone, bursicon. This is supported by evidence that wing cell death is inhibited by a mutation of Rickets, which encodes a G-protein coupled receptor in the glycoprotein hormone family that is a putative bursicon receptor. Furthermore, stimulation of components downstream of bursicon, such as a membrane permeable analog of cAMP, or ectopic expression of constitutively active forms of G proteins or PKA, induces precocious death. Conversely, cell death is inhibited in wing clones lacking G protein or PKA function. Thus, activation of the cAMP/PKA signaling pathway is required for transduction of the hormonal signal that induces wing epidermal cell death after eclosion (Kimura, 2004).

To determine whether a humoral signal coming from the head triggers cell death, the necks of flies were ligated at various times after eclosion and the wings were examined for cell death at 2 hours after the ligation. Ligation just after eclosion suppresses cell death and GFP was still detectable in the nuclei of wing epidermal cells after 2 hours. By contrast, when flies were ligated at 20 minutes after eclosion, the normal pattern of cell death was observed. Ligation at later stages correlates with an increased percentage of flies with wing epidermal cell death. Thus wing epidermal cell death is triggered by a signal emanating from the head shortly after eclosion (Kimura, 2004).

The cellular effects of cAMP are usually mediated by PKA. To determine whether this is also the case for wing epidermal cell death, the effect of reduction or elimination of PKA activity on cell death was examined. A dominant-negative form of the regulatory subunit of PKA (R*), whose ectopic expression is known to reduce the activity of endogenous PKA, was used. When R* was ectopically expressed using the en-Gal4 driver, many cells of the wings remained at 2 hours, or even at 8 hours, after wing spreading, resulting in separation between the ventral and dorsal cuticular sheets in the posterior compartment. Targeted expression of R* caused wavy or curly wings, probably due to the distortion between normal adhesion of dorsoventral cuticles in the anterior compartment and detachment of the cuticle in the posterior compartment (Kimura, 2004).

Next, DC0-dependent PKA activity was eliminated by generating clones of DC0 mutant cells within the developing wings. The DC0 gene in Drosophila encodes a catalytic subunit, one of the components of PKA. Since clones of DC0 mutant cells in the anterior compartment produce anterior duplication of the normal wing pattern, the clones in the posterior compartment were examined to investigate whether the death of wing epidermal cells marked with Histone-GFP is suppressed or not. The cells of the clones remained at 2 hours after wing spreading, although the surrounding cells had already been eliminated by cell death. Thus, reduction or elimination of PKA activity prevents the death of wing epidermal cells (Kimura, 2004).

The effects of constitutive activation of PKA on cell death were examined. A mutationally altered mouse catalytic subunit (mC*) was used that is resistant to inhibition by the regulatory subunit. The mutant catalytic subunit is constitutively active, irrespective of cAMP concentration, and can function in Drosophila cells. Using the en-Gal4 driver, the constitutively active catalytic subunit of mC* was expressed in wing epidermal cells. All eclosing flies had blistered wings. The wing epidermal cells died prior to wing spreading. Thus, constitutive activation of PKA causes the precocious death of wing epidermal cells (Kimura, 2004).

The induction of cell death was examined at various stages of pharate adults. As seen in the cases of cAMP injection and of ectopic expression of Gs{alpha}*, precocious cell death was induced at G stage and later. This indicates that wing cells acquire competence to respond to PKA activity by G stage, about 3 hours before eclosion (Kimura, 2004).

A mutation in the G-protein coupled receptor gene rickets inhibits wing epidermal cell death. In Drosophila, the rickets gene is a member of the glycoprotein hormone receptor family of the G-protein-coupled receptors and has been suggested to encode a bursicon receptor. Wing epidermal cell death, marked by Histone-GFP, was examined in rk mutants. In the mutants, wing epidermal cells remained at 2 hours, or even at 8 hours, after eclosion. To determine whether the inhibition of cell death is caused by a failure in the reception of a hormonal signal inducing death, the effects were examined of 8-Br-cAMP and hemolymph injection into rk mutants that were neck-ligated at eclosion. In wild-type flies, injection of hemolymph from wild-type flies at 30 minutes after eclosion and injection of 8-Br-cAMP induces cell death. However, in rk mutants, cell death was induced by injection of 8-Br-cAMP but not by injection of hemolymph. This indicates that the mutant cells could not receive the hormonal signal in the hemolymph, although the activation of cell death by cAMP/PKA signaling was normal in the mutant cells (Kimura, 2004).

The peptide hormone, bursicon, is known to play a role in the post-ecdysial phase of development. Bursicon has been shown to be released before wing expansion and to hasten the tanning reaction, serving to harden the newly expanded cuticle. The results of this study suggest that the hormone that induces cell death of the wing epidermis could be bursicon. (1) Neck ligation and hemolymph injection experiments demonstrated that the triggering signal to induce death is a humoral factor released after eclosion. This temporal pattern of death-inducing activity in the hemolymph corresponds to that of bursicon. (2) Injection of cAMP induced cell death, implicating cAMP as the second messenger in the cell death pathway. Studies in blowflies have shown that bursicon also acts through cAMP. Recently, in Drosophila, cAMP was shown to induce cuticular melanization in a fashion similar to bursicon. (3) Reception of the hormonal signal inducing cell death is mediated by a probable bursicon receptor, Rickets (DLGR2), which also acts through cAMP. (4) In Lucilia cuprina, it has been proposed that bursicon is the same as fragment disaggregating hormone, which increases the circulating filamentous cellular fragments derived from post-ecdysial death of the wing epidermal cells. Taken together, it is likely that bursicon coordinates events such as the cell death of wing epidermis and the subsequent tanning and hardening of the cuticle. However, another possibility cannot be ruled out -- namely, that several humoral factors could signal through the pathway. Identification of a bursicon gene as CG13419 in Drosophila will facilitate genetic approaches to understand the role of bursicon in wing epidermal cell death (Kimura, 2004).

The Drosophila DCO mutation suppresses age-related memory impairment (AMI) without affecting lifespan: AMI is caused by an age-related disruption of amnesiac-dependent memory via PKA activity in mushroom bodies

The study of age-related memory impairment (AMI) has been hindered by a lack of AMI-specific mutants. In a screen for such mutants in Drosophila, it was found that heterozygous mutations of DCO (DCO/+), which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), delay AMI more than twofold without affecting lifespan or memory at early ages. AMI is restored when a DCO transgene is expressed in mushroom bodies, structures important for olfactory memory formation. Furthermore, increasing cAMP and PKA activity in mushroom bodies causes premature AMI, whereas reducing activity suppresses AMI. In Drosophila AMI consists of a specific reduction in memory dependent on the amnesiac (amn) gene. amn encodes putative neuropeptides that have been proposed to regulate cAMP levels in mushroom bodies. Notably, both the memory and AMI defects of amn mutants are restored in amn;DCO/+ double mutants, suggesting that AMI is caused by an age-related disruption of amn-dependent memory via PKA activity in mushroom bodies (Yamazaki, 2007).

The molecular mechanisms affecting both AMI and aging are likely to be tightly linked. In general, mechanisms and mutations that extend lifespan also delay the onset of AMI. For example, calorie restriction both increases longevity and improves memory in aged animals. Ames dwarf mice, which have reduced growth hormone, thyroid-stimulating hormone and prolactin, live longer than their wild-type siblings and show delayed age-related declines in performance of an inhibitory avoidance task. Furthermore, antioxidant activity has been associated with both extension of lifespan and delayed AMI (Yamazaki, 2007).

There is also evidence that aging and AMI can be separated, however. The optimal level of calorie restriction required for maximal extension of lifespan is not necessarily optimal for delaying AMI, and signaling by growth hormone and insulin-like growth factor activity, which promotes aging, ameliorates AMI. Thus, although it is likely that aging and AMI share pathways in common, at some point these pathways diverge (Yamazaki, 2007).

In Drosophila, genetic studies of memory after a single cycle of training in a Pavlovian olfactory association task have revealed four temporal memory phases: initial learning (LRN), short-term memory (STM), middle-term memory (MTM) and anesthesia-resistant memory (ARM). These phases have been shown to be distinct based on the existence of mutants and pharmacological sensitivities that are specific to particular memory phases. To study how aging affects these phases, AMI has been characterized in Drosophila, and it has been determined that aging specifically impairs MTM, which is dependent on the function of the amn gene (Tamura, 2003). Memory defects in aged flies become identical to those of amn mutants and amn mutants do not show any memory decay upon aging (Yamazaki, 2007).

However, amn expression does not decrease upon aging and overexpression of an amn transgene does not ameliorate AMI. The amn gene products are proposed to be putative neuropeptides secreted from two dorsal-paired medial (DPM) neurons that innervate the mushroom bodies, neural centers for learning and memory. Therefore, it was theorized that an age-related disruption of signaling downstream of amn leads to memory impairment. This suggests that AMI may arise in the mushroom bodies, and led to a screen for genes regulating AMI in the mushroom bodies (Yamazaki, 2007).

In the present study identified heterozygous mutations in DCO (also called Pka-C1), the gene encoding the catalytic subunit of PKA, as strong AMI suppressors. Notably, heterozygous mutations in DCO affect neither memory at early ages nor lifespan, suggesting that DCO hypomorphs are AMI-specific mutants. PKA activity in the mushroom bodies mediates an age-related decline in amn-dependent MTM (Yamazaki, 2007).

From a screen of 54 fly lines with mutations in genes expressed predominantly in mushroom bodies, two lines were identified with altered AMI. Among these, heterozygous mutations in DCO function as strong AMI suppressors. Although it has been shown that mutations and conditions that extend lifespan tend also to delay AMI, the DCO mutations seem to function by a different mechanism. In contrast to low temperature and calorie restriction, which seem to extend lifespan and ameliorate AMI proportionately, DCO/+ mutants have no effect on lifespan but delay AMI onset and severity to much greater extents than manipulations that alter lifespan. Thus, it is envisioned that these mutants lie downstream of aging pathways and specifically suppress the negative effects that aging has on memory (Yamazaki, 2007).

Decreasing cAMP and PKA activity by using four distinct heterozygous DCO mutations or by expressing PKI significantly delays AMI. Increasing cAMP and PKA activity either by using a heterozygous dnc1 mutant or by overexpressing DCO results in disruption of memory reminiscent of premature AMI. In addition, AMI is restored in DCOB3/+ flies when DCO is expressed in the mushroom bodies but not when it is expressed in the fan-shaped body or ellipsoid body. From these data, it is concluded that cAMP and PKA signaling activity in the mushroom bodies is a cause of AMI (Yamazaki, 2007).

Several PKA substrates have been associated with AMI. For example, calcium dysregulation caused by increased activity of the L-type voltage-gated calcium channel (LVGCC) Cav1.2 has been proposed to cause AMI. Notably, LVGCC expression is highly upregulated in aging and channel activity is strongly enhanced by PKA phosphorylation, suggesting that LVGCCs may be a candidate substrate that can cause AMI during aging. In support of this hypothesis, LVGCC phosphorylation has been shown to be increased in the hippocampus of aged rats. Phosphorylated tau protein is a major component of neurofibrillary tangles (NFT), and accumulation of NFTs shows strong correlation with age-related memory loss. PKA is one of several kinases that phosphorylate tau protein and facilitate NFT formation (Yamazaki, 2007).

AMI results from a specific reduction of amn-dependent MTM, and amn mutants show memory and AMI defects reminiscent of the phenotype of flies overexpressing DCO+. Both these memory and AMI defects are suppressed in amn;DCO/+ double mutants, strongly suggesting that amn and DCO function in a common pathway and DCO function is downstream of amn function. The amn gene encodes at least three peptides, two of which have homologies to mammalian pituitary adenylyl cyclase-activating peptide (PACAP) and growth hormone-releasing hormone (GHRH). In mammalian systems, receptors of PACAP and GHRH function to increase adenylyl cyclase activity. At the Drosophila neuromuscular junction, amn mutations cause decreases in Ca2+ currents through LVGCCs as a result of decreased PKA activity. These results support the idea that Amn peptides stimulate adenylyl cyclase activity. It has also been reported, however, that Ca2+ currents through LVGCCs in the mushroom bodies are greatly enhanced by amn mutations. Thus amn mutants seem to have opposite phenotypes at the neuromuscular junction and in the mushroom bodies. Although it remains possible that amn functions differently at these two locations, it is hypothesized that, instead, indirect effects may occur in the mushroom bodies of amn mutants that increase intracellular Ca2+ concentrations. A drastic increase in Ca2+ influx may enhance activity of the Ca2+- and calmodulin-dependent adenylyl cyclase rutabaga, leading to aberrant increases in activity-dependent PKA activity that can be suppressed by DCO/+ mutations (Yamazaki, 2007).

It has been reported that inhibition of cAMP and PKA activity in the prefrontal cortex (PFC) improves working memory in aged, cognitively impaired rats but not in young rats, whereas activation of cAMP and PKA impairs memory in aged rats at lower concentrations than in young rats. Basal levels of PKA, and of several adenylyl cyclase and phosphodiesterase isoforms, do not show age-related changes in the aged rat PFC, a finding similar to results obtained from flies. CREB phosphorylation, which is likely to be downstream of cAMP/PKA signaling, is increased in the aged rat PFC. These results suggest that several aspects of AMI are conserved between Drosophila and mammals and support a model in which increasing PKA-dependent phosphorylation at a post-translational step may be responsible for AMI (Yamazaki, 2007).

As PKA activity is essential for memory formation, it seems counterintuitive that PKA is also responsible for an age-dependent memory reduction. Indeed, in mammalian systems, it has been widely reported that increasing cAMP levels can enhance both the protein synthesis-dependent phase of hippocampal long-term potentiation (LTP) and hippocampus-dependent long-term memory in aged mice. These results have been used to suggest that an age-dependent reduction in cAMP and PKA activity may cause AMI. However, increasing cAMP improves memory in young as well as old mice, indicating that this is a general rather than an age-specific effect (Yamazaki, 2007).

If activation of the cAMP/PKA pathway improves hippocampal memory and LTP, why isn't the activity of this pathway higher in the wild-type organism? Although speculative, it seems likely that cAMP/PKA activity must have adverse effects, preventing high PKA expression. The current data are consistent with a model in which acute PKA activity is required for memory but long-term effects of PKA promote AMI. Thus, the levels of PKA observed naturally may be the result of a balance between two antagonistic pleiotropic effects of PKA (Yamazaki, 2007).

Squid. identified in a screen for modifiers of the Protein Kinase A oogenesis polarity phenotype, is required for the establishment of anteroposterior polarity in the Drosophila oocyte

The heterogeneous nuclear ribonucleoprotein (hnRNP) Squid (Sqd) is a highly abundant protein that is expected to bind most cellular RNAs. Nonetheless, Sqd plays a very specific developmental role in dorsoventral (DV) axis formation during Drosophila oogenesis by localizing gurken (grk) RNA. This study reports that Sqd is also essential for anteroposterior (AP) axis formation. sqd was identified in a screen for modifiers of the Protein Kinase A (PKA) oogenesis polarity phenotype. The AP defects of sqd mutant oocytes resemble those of PKA mutants in several ways. In both cases, the cytoskeletal reorganization at mid-oogenesis, which depends on a signal from the posterior follicle cells, does not produce a correctly polarized microtubule (MT) network. This causes the posterior determinant, oskar (osk) RNA, to localize to central regions of the oocyte, where it is ectopically translated. Additionally, MT-dependent anterior movement of the oocyte nucleus and the grk-dependent specification of posterior follicle cells are unaffected in both mutants. However, in contrast to PKA mutants, sqd mutants do not retain a discrete posterior MT organizing center (MTOC) capable of supporting ectopic posterior localization of bicoid (bcd) RNA. sqd mutants also display several other phenotypes not seen in PKA mutants; these probably result from the disruption of MT polarity in earlier stages of oogenesis. Loss of Sqd does not affect polarity in follicle cells, wings or eyes, indicating a specific role in the determination of MT polarity within the germline (Steinhauer, 2005).

It is well established that the hnRNP Sqd participates in Drosophila DV axis formation. Following a screen to identify factors that interact with PKA in mid-oogenesis, Sqd was discovered to be essential also for AP axis formation. The localization of posterior factors, including osk RNA, GFP-Stau, kin-ß-gal, and Dhc, was disrupted at stages 9-10 in all sqd allelic combinations tested, was highly penetrant in strong sqd alleles, and could be rescued by expression of a Sqd cDNA transgene. These defects can be attributed to the failure of sqd mutants to establish a normally polarized MT array at mid-oogenesis. Defects were also observed in MT organization and in the localization of posterior factors, including Grk protein, in sqd mutant oocytes at stages 2-6. Despite the imperfect localization of Grk before stage 6, posterior fate appears to be specified normally in the follicle cells overlying sqd mutant oocytes. Thus, sqd mutations affect germline-specific processes required for the polarization of MTs in both early and mid-oogenesis (Steinhauer, 2005).

Loss of PKA in the germline does not affect MT polarity in early oogenesis, as judged by Orb localization in stages 2-6, but, similar to sqd mutants, it disrupts MT polarity in mid-oogenesis without discernibly altering follicle cell fate. However, despite several similarities, a significant difference was observed in the MT organization of sqd and PKA mutants at stages 8-10 (Steinhauer, 2005).

In grk and cornichon (cni ) mutants, where the posterior follicle cells do not differentiate properly, both the subsequent RNA localization defects and the failure of the oocyte nucleus to migrate from the posterior to the anterior have been attributed to defects in MT reorganization resulting from loss of a posterior follicle cell signal. In PKA and sqd mutant oocytes, the anterior migration of the oocyte nucleus, which depends on MT function, is unaffected, despite the accompanying MT defects and the highly penetrant mislocalization of osk RNA and other posterior factors. Thus, it appears that either discrete aspects of the MT organization, which direct nucleus migration, are spared in PKA and sqd mutants or the overall disruption of MT organization by loss of PKA or Sqd is simply less severe than that caused by loss of posterior follicle cell fate (Steinhauer, 2005).

The normal organization of MTs in stage 8-10 oocytes is not entirely clear. In addition to MTs nucleated at the anterior cortex, MTs have been proposed to emanate from all cortical positions, with the exception of the posterior pole. This assertion is based on the observations that components associated with MT minus-ends, such as gamma-tubulin and the centrosome component Centrosomin (Cnn), can be seen along the entire oocyte cortex, and that injected bcd RNA localizes to the lateral cortices as well as the anterior, but not to the posterior pole. Hence, normal posterior localization of osk RNA may require the clearing of MTs nucleated both from a discrete posterior MTOC established before stage 6 and from dispersed cortical sites established after stage 7 (Steinhauer, 2005).

Staining with alpha-tubulin antibody following partial MT depolymerization revealed MT stubs emanating mostly from the anterior in wild-type oocytes, whereas PKA and sqd mutant oocytes retain short MTs around the entire oocyte cortex, including the posterior pole. Some PKA mutant oocytes also show an elevated posterior concentration of MTs not seen in sqd mutant oocytes. Thus, it appears that the primary MT defect in sqd mutants is the failure to eliminate cortical sites of MT nucleation beyond stage 7, whereas PKA mutants additionally retain a posterior MTOC beyond stage 6. This hypothesis can explain why ectopic bcd RNA localizes at the posterior of PKA mutant oocytes but not sqd mutant oocytes. It should, however, be noted that since classical MTOC components, such as gamma-tubulin, are present along the entire oocyte cortex at stages 9-10 even in wild-type oocytes, the inference of a discrete posterior MTOC from partial MT depolymerization experiments cannot be confirmed directly (Steinhauer, 2005).

In a proportion of sqd mutant stage 2-6 oocytes, Grk, Orb, osk RNA and MTs are distributed evenly throughout the ooplasm rather than localizing in a cap at the oocyte posterior. Although these defects do not appear to cause the subsequent AP defects by preventing posterior follicle cell specification, the possibility cannot be ruled out that the early and late polarity phenotypes are causally related in some other way. For instance, it is possible that a molecule(s) required at the posterior of the oocyte for the MT reorganization at stages 7-8 is improperly localized by stage 6 in sqd mutants, as are Grk, Orb and osk RNA. If MT rearrangements are very sensitive to the localized concentration of this hypothetical regulator, an early polarity defect of apparently low penetrance could be translated into a much more penetrant polarity phenotype at mid-oogenesis (Steinhauer, 2005).

sqd is not the only mutant to cause polarity defects in both early and mid-oogenesis. For example, defects in early polarity are caused by mutations in Armitage (Armi), a component of the RNA silencing machinery, and these defects have been proposed to be the cause of a mid-oogenesis AP polarity phenotype. However, it was found that pnt998/12 expression is not disrupted in armi1 homozygotes. Weak par-1 alleles also affect mid-oogenesis polarity without affecting posterior follicle cell fate, whereas strong alleles disrupt early polarity severely, causing oocyte identity to be lost. Thus, for several mutations, including sqd, armi and par-1, it is unclear whether MT organization is disrupted independently at two distinct phases of development or whether there is a causal connection between the early and later polarity phenotypes that is not evident as a failure in posterior follicle cell specification. In either case, a single molecular target might account for both the early and mid-oogenesis phenotypes (Steinhauer, 2005).

Several additional phenotypes became prevalent in older sqd j4B4 germline clones, rising to very high penetrances after 2 weeks. Among the varied late onset sqd phenotypes, the oocyte sometimes was mispositioned within the egg chamber, even in those egg chambers containing the normal complement of nurse cells to oocyte. This phenotype can arise in several ways, including as a result of delayed oocyte specification. A role for Sqd in oocyte specification is supported by the presence of cysts with 16 nurse cells and no oocyte in these older ovarioles. In other cases, cysts with fewer than 16 germ cells were observed, implicating Sqd in cystocyte mitosis. Both oocyte specification and the normal cystocyte divisions depend on specific arrangements of the MT cytoskeleton in the germarium. Thus, it is likely that some of these late onset sqd phenotypes, like the polarity phenotypes, are caused by a defect in regulating MT dynamics (Steinhauer, 2005).

In sqd j4B4 germline clones, the accumulation of Osk protein was noticed in the cytoplasm of stage 9-10 oocytes. A similar observation was reported for mutations in another hnRNP A/B family member, Hrp48 (Hrb27C - FlyBase) (Yano, 2004). Normally, Osk protein accrues only at the posterior cortex, and translation of osk RNA is presumably repressed elsewhere. Therefore, loss of sqd may cause de-repression of osk translation. However, no ectopic osk translation was seen in stage 6-8 sqd mutant oocytes, detected either with Osk antibody or with an osk translation reporter, in contrast to the premature expression observed with the osk translation reporter in hrp48 mutants (Yano, 2004) or with similar reporters lacking specific repressor elements. Thus, although the idea that sqd is directly involved in translational regulation of osk cannot be dismissed, an alternative hypothesis is proposed for the ectopic Osk protein accumulation in sqd mutants. Since most of the posterior components examined were mislocalized to the center of sqdj4B4 oocytes, it is believed that the primary AP defect in sqd mutants is that the MT plus-ends are focused incorrectly at the center of the oocyte. Hence, all the necessary components for osk translation may be localized together, and it is hypothesized that the osk translation machinery is assembled and activated in the middle of the sqd mutants as it normally is at the posterior of wild-type oocytes at stage 9 (Steinhauer, 2005).

A low penetrance of ectopic Osk protein was detected in PKA mutants. The scenario outlined above could be true for PKA mutants as well. Regardless of the mechanism, it is clear from this result that PKA is not absolutely required for Osk translation, although it may enhance osk translation (Steinhauer, 2005).

Although sqd was identified in a screen for modifiers of PKA in oocyte polarity, retesting with various alleles indicated that there is not a strong genetic interaction between the two loci. Both Sqd and PKA act in mid-oogenesis to reorganize the oocyte MTs in response to a normal posterior follicle cell signal, but specific MT defects differ between the two mutants. Thus, they probably have different targets and mechanisms in this complex process (Steinhauer, 2005).

The hnRNP Sqd is an RNA-binding protein. Another hnRNP of the same family, Hrp48, is also required for MT reorganization at mid-oogenesis (Yano, 2004). Sqd and Hrp48 bind each other in vitro, cooperate in grk RNA localization and have similar localization patterns throughout oogenesis. Thus, one might expect these two proteins to act together in MT reorganization. Although no strong genetic interaction was detected between sqd and hrp48 in AP polarity, it is speculated that they are collectively necessary for the localization and translation of one or a small number of specific RNA molecules required for MT repolarization at mid-oogenesis (Steinhauer, 2005).

hnRNPs normally participate in the processing of many RNAs, but their generic functions may be partially redundant, so that, for example, in sqd mutants, continued cell viability is not impaired despite the presence of a strong AP polarity defect. The ability to induce large, persistent somatic cell clones for sqd j4B4 without causing any polarity or other phenotypes supports this idea. Follicle cell polarity was also normal in PKA mutant clones. Thus, the disruptions in MT polarity that were observed for both PKA and sqd mutants represent specialized functions of these proteins in germline cells (Steinhauer, 2005).

Retrograde signaling by Syt 4, involving activation of the presynaptic PKA pathway, in induces presynaptic release and synapse-specific growth

The molecular pathways involved in retrograde signal transduction at synapses and the function of retrograde communication are poorly understood. Postsynaptic calcium 2+ ion (Ca2+) influx through glutamate receptors and subsequent postsynaptic vesicle fusion trigger a robust induction of presynaptic miniature release after high-frequency stimulation at Drosophila neuromuscular junctions. An isoform of the synaptotagmin family, Synaptotagmin 4 (Syt 4), serves as a postsynaptic Ca2+ sensor to release retrograde signals that stimulate enhanced presynaptic function through activation of the cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase pathway. Postsynaptic Ca2+ influx also stimulates local synaptic differentiation and growth through Syt 4-mediated retrograde signals in a synapse-specific manner (Yoshihara, 2005).

Neuronal development requires coordinated signaling to orchestrate pre- and post-synaptic maturation of synaptic connections. Synapse-specific enhancement of synaptic strength as occurs during long-term potentiation, as well as compensatory homeostatic synaptic changes, have been suggested to require retrograde signals for their induction. Although retrograde signaling has been implicated widely in synaptic plasticity, the molecular mechanisms that transduce postsynaptic Ca2+ signals during enhanced synaptic activity to alterations in presynaptic function are poorly characterized. Because postsynaptic Ca2+ is essential for synapse-specific potentiation, it is important to characterize how Ca2+ can regulate retrograde communication at synapses (Yoshihara, 2005).

To dissect the mechanisms underlying activity-dependent synaptic plasticity, tests were performed to see whether newly formed Drosophila glutamatergic neuromuscular junctions (NMJs), which have ~30 active zones, show physiological changes after 100-Hz stimulation. Within 1 min after stimulation, a gradual 100-fold increase in miniature excitatory postsynaptic current (miniature) frequency was observed, from a baseline of 0.03 Hz to often more than 5 Hz. The high-frequency-stimulation-induced miniature release (termed HFMR) continued for a few minutes to as long as 20 min before subsiding to baseline levels. Perfusion of postsynaptic muscles with the Ca2+ chelator EGTA from the patch pipette caused a modest suppression of HFMR, whereas the fast Ca2+ chelator BAPTA induced strong suppression by 2.5 min of perfusion. Longer perfusion with BAPTA for 5 min before stimulation abolished HFMR, indicating HFMR is induced after postsynaptic Ca2+ influx (Yoshihara, 2005).

Ca2+-induced vesicle fusion in presynaptic terminals provides a temporally controlled and spatially restricted signal essential for synaptic communication. Postsynaptic vesicles within dendrites have been visualized by transmission electron microscopy, and dendritic release of several neuromodulators has been reported. To test whether postsynaptic vesicle fusion might underlie the Ca2+-dependent release of retrograde signals, postsynaptic vesicle recycling was blocked by using the dominant negative shibirets1 mutation, which disrupts endocytosis at elevated temperatures. shibirets1 was expressed specifically in postsynaptic muscles by driving a UAS-shibirets1 transgene with muscle-specific myosin heavy chain (Mhc)-Gal4, keeping presynaptic activity intact. At the permissive temperature (23°C), high-frequency stimulation induced normal HFMR. However, raising the temperature to 31°C suppressed HFMR in the presence of postsynaptic shibirets1, whereas wild-type animals displayed normal HFMR at 31°C. Basic synaptic properties in Mhc-Gal4, UAS-shibirets1 animals were not affected at either the permissive or the restrictive temperature. The suppression of HFMR is not due to irreversible damage induced by postsynaptic UAS-shibirets1 expression, because a second high-frequency stimulation after recovery to the permissive temperature induced normal HFMR (Yoshihara, 2005).

The synaptic vesicle protein synaptotagmin 1 (Syt 1) is the major Ca2+ sensor for vesicle fusion at presynaptic terminals but is not localized postsynaptically. Another isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), is present in the postsynaptic compartment (Adolfsen, 2004), suggesting Syt 4 might function as a postsynaptic Ca2+ sensor. Syt 4 immunoreactivity is observed in a punctate pattern surrounding presynaptic terminals, suggesting Syt 4 is present on postsynaptic vesicles. Postsynaptic vesicle recycling was again blocked by using the UAS-shibirets1 transgene driven with Mhc-Gal4. Without a temperature shift, Syt 4-containing vesicles show their normal postsynaptic distribution surrounding presynaptic terminals. When the temperature is shifted to 37°C for 10 min in the presence of high-K+ saline containing 1.5 mM Ca2+ to drive synaptic activity, Syt 4-containing vesicles translocate to the plasma membrane. After recovery at 18°C for 20 min, postsynaptic vesicles return to their normal position. Removing extracellular Ca2+ during the high-K+ stimulation results in vesicles that do not translocate to the postsynaptic membrane (Yoshihara, 2005).

To further test whether the Syt 4 vesicle population undergoes fusion with the postsynaptic membrane as opposed to mediating fusion between intracellular compartments, transgenic animals were constructed expressing a pH-sensitive green fluorescent protein (GFP) variant (ecliptic pHluorin) fused at the intravesicular N terminus of Syt 4. Ecliptic pHluorin increases its fluorescence 20-fold when exposed to the extracellular space from the acidic lumen of intracellular vesicles during fusion. Expression of Syt 4-pHluorin in postsynaptic muscles resulted in intense fluorescence at specific subdomains in the postsynaptic membrane, defining regions where Syt 4 vesicles undergo exocytosis. The fluorescence was not diffusely present over the postsynaptic membrane but directed to restricted compartments. Mhc-Gal4, UAS-Syt 4-pHluorin larvae were co-stained with antibodies against the postsynaptic density protein, DPAK, and nc82, a monoclonal antibody against a presynaptic active zone protein. Syt 4-pHluorin colocalized with DPAK and localized adjacent to nc82, demonstrating that Syt 4-pHluorin translocates from postsynaptic vesicles to the plasma membrane at postsynaptic densities opposite presynaptic active zones (Yoshihara, 2005).

To examine the function of Syt 4-dependent postsynaptic vesicle fusion, the phenotype of a syt 4 null mutant (syt 4BA1) ( Adolfson, 2004) and a syt 4 deficiency (rn16) was characterized. Mutants lacking Syt 4 hatch from the egg case 21 hours after egg laying at 25°C, similar to wild type, and grow to fully mature larvae that pupate and eclose with a normal time course. To determine whether postsynaptic vesicle fusion triggered by Ca2+ influx is required for HFMR, the effects of high-frequency stimulation were analyzed in syt 4 mutants. In contrast to controls, the increase of miniature release was eliminated in syt 4 mutants. Postsynaptic expression of a UAS-syt 4 transgene completely restored HFMR in the null mutant, demonstrating that postsynaptic Syt 4 is required for triggering enhanced presynaptic function. Presynaptic expression of a UAS-syt 4 transgene did not restore HFMR. In addition, postsynaptic expression of a mutant Syt 4 with neutralized Ca2+-binding sites in both C2A and C2B domains did not rescue HFMR, indicating that retrograde signaling by Syt 4 requires Ca2+ binding (Yoshihara, 2005).

The large increase in miniature frequency observed during HFMR is similar to the enhancement of presynaptic release after activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) described in Aplysia and Drosophila. Bath application of forskolin, an activator of adenylyl cyclase, results in a robust enhancement of miniature frequency at Drosophila NMJs similar to that observed during HFMR, suggesting retrograde signals may function to increase presynaptic cAMP. To test the role of the cAMP-PKA pathway in HFMR, DC0 mutants were assayed for the presence of HFMR. DC0 encodes the major catalytic subunit of PKA in Drosophila and has been implicated in olfactory learning. Similar to the lack of forskolin-induced miniature induction, DC0 null mutants lack HFMR. Bath application of forskolin in syt 4 mutants resulted in enhanced miniature frequency, suggesting activation of the cAMP pathway can bypass the requirement for Syt 4 in synaptic potentiation (Yoshihara, 2005).

To further explore the role of retrograde signaling at Drosophila synapses, the role of activity in synapse differentiation and growth was characterized. During Drosophila embryonic development, presynaptic terminals undergo a stereotypical structural change from a flat path-finding growth cone into varicose synaptic terminals through dynamic reconstruction. Such developmental changes in synaptic structure may share common molecular mechanisms with morphological changes induced during activity-dependent plasticity. Synaptic transmission was eliminated by using a deletion mutation that removes the postsynaptic glutamate receptors, DGluRIIA and DGluRIIB (hereafter referred to as GluRs). Postsynaptic currents normally induced by nerve stimulation were completely absent in the mutants (gluR). Miniatures were also eliminated, even at elevated extracellular Ca2+ concentrations of 4 mM. In the absence of GluRs, the presynaptic morphology of motor terminals is abnormal, even though GluRs are only expressed in postsynaptic muscles. GluR-deficient terminals maintain a flattened growth cone-like structure and fail to constrict into normal synaptic varicosities. Synaptic development was assayed in a null mutant of the presynaptic plasma membrane t-SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) receptor], syntaxin (syx), which eliminates neurotransmitter release, providing an inactive synapse similar to that in the gluR mutant. syx null mutants also have abnormal growth cone-like presynaptic terminals with less varicose structure (Yoshihara, 2005).

Because activity is required for synapse development, whether Syt 4-dependent vesicle fusion may be required, similar to its role in acute retrograde signaling during HFMR, was tested. Physiological analysis revealed that the amplitude of evoked currents in mutants lacking Syt 4 was moderately reduced compared with wild type, suggesting weaker synaptic function or development. Similar to the morphological phenotype of the gluR mutant, syt 4 null mutant embryos show defective presynaptic differentiation. Nerve terminals lacking Syt 4 display reduced varicose structure, whereas wild-type terminals have already formed individual varicosities at this stage of development. Postsynaptic expression with a UAS-syt 4 transgene rescues the physiological and morphological phenotypes. Syt 4 Ca2+-binding deficient mutant transgenes did not rescue either the morphological immaturity or the reduced amplitude of evoked currents, even though Syt 4 immunoreactivity at the postsynaptic compartment was restored by muscle-specific expression of the mutant syt 4 transgene, similar to the wild-type syt 4 transgene and endogenous Syt 4 immunoreactivity (Yoshihara, 2005).

Mammalian syt 4 was originally identified as an immediate-early gene that is transcriptionally up-regulated by nerve activity in certain brain regions (Vician, 1995). Therefore, gain-of-function phenotypes caused by postsynaptic Syt 4 overexpression were examined specifically in muscle cells to increase the probability of postsynaptic vesicle fusion. Syt 4 overexpression induced overgrowth of presynaptic terminals in mature third instar larvae, in contrast to overexpression of Syt 1, which does not traffic to Syt 4-containing postsynaptic vesicles. In addition to synaptic overgrowth, Syt 4 overexpression occasionally induced the formation of abnormally large varicosities. Postsynaptic overexpression of the Syt 4 Ca2+-binding mutant did not induce synaptic overgrowth, indicating that retrograde signaling by Syt 4 also requires Ca2+ binding to promote synaptic growth (Yoshihara, 2005).

To determine whether the cAMP-PKA pathway is important in activity-dependent synaptic growth, the effects of PKA on synaptic morphology were assayed. Expression of constitutively active PKA presynaptically using a motor neuron-specific Gal4 driver induced not only synaptic overgrowth but also larger individual varicosities in mature third instar larvae, similar to those induced by postsynaptic overexpression of Syt 4. These observations are consistent with the presynaptic overgrowth observed in the learning mutant, dunce, which disrupts the enzyme that degrades cAMP, and with studies in Aplysia implicating PKA in synaptic varicosity formation. The loss-of-function phenotype of PKA mutants (DC0B3) was characterized at the embryonic NMJ, to compare with gluR and syt 4 mutants. Presynaptic terminals in the DC0 mutant were morphologically aberrant, with abnormal growth cone-like features and less varicose structure. Postsynaptic expression of a constitutively active PKA transgene in the DC0 or syt 4 mutant backgrounds rescued the immature morphology, suggesting activation of PKA is downstream of Syt 4-dependent release of retrograde signals (Yoshihara, 2005).

Similar to the role of Syt 1-dependent synaptic vesicle fusion in triggering synaptic transmission at individual synapses, Syt 4-dependent vesicle fusion might trigger synapse-specific plasticity and growth. To test synapse specificity, advantage was taken of the specific properties of the Drosophila NMJ at muscle fibers 6 and 7, where two motorneurons innervate both muscle fibers 6 and 7 during development. Syt 4 was expressed specifically in embryonic muscle fiber 6 but not muscle fiber 7 by using the H94-Gal4 driver. If Syt 4-dependent retrograde signals induce general growth of the motorneuron, one would expect to see a proliferation of synapses on both muscle fibers. Alternatively, if Syt 4 promoted local synaptic growth, one would expect specific activation of synapse proliferation only on target muscle 6, releasing the Syt 4-dependent signal. UAS-syt 4 driven by H94-Gal4 increased innervation on muscle fiber 6 compared with that on muscle fiber 7 in third instar larvae. Control experiments with Syt 4 Ca2+-binding deficient mutant transgenes, or a transgene encoding Syt 1, did not result in proliferation. Thus, synaptic growth can be preferentially directed to specific postsynaptic targets where Syt 4-dependent retrograde signals predominate, allowing differential strengthening of active synapses via local rewiring (Yoshihara, 2005).

On the basis of these results, a local feedback model is proposed for activity-dependent synaptic plasticity and growth at Drosophila NMJs. Synapse-specific Ca2+ influx triggers postsynaptic vesicle fusion through Syt 4. Fusion of Syt 4-containing vesicles with the postsynaptic membrane releases locally acting retrograde signals that activate the presynaptic terminal, likely through the cAMP pathway. Active PKA then triggers cytoskeletal changes by unknown effectors to induce presynaptic growth and differentiation. Moreover, PKA is well known to facilitate neurotransmitter release directly, triggering a local synaptic enhancement of presynaptic release as shown in HFMR. Therefore, postsynaptic vesicular fusion might initiate a positive feedback loop, providing a localized activated synaptic state that can be maintained beyond the initial trigger (Yoshihara, 2005).

As a general mechanism for memory storage, Hebb postulated that potentiated synapses maintain an activated state until structural changes occur to consolidate alterations in synaptic strength (Hebb, 1949). The current results demonstrate that acute plasticity and synapse-specific growth require Syt 4-dependent retrograde signaling at Drosophila NMJs. The feedback mechanism described here could be a molecular basis for both input-specific postsynaptic tagging and an output-specific presynaptic mark or tag for long-lasting potentiation. The regenerative nature of a positive feedback signal allows individual synapses to be tagged in a discrete all-or-none manner until synaptic rewiring is completed. The synaptic tag is maintained as a large increase in miniature frequency at Drosophila NMJs, suggesting a previously unknown role for miniature release in neuronal function. The spatial resolution for input and output specificity would result from the accuracy insured by Ca2+-dependent vesicle fusion and subsequent diffusion, similar to the precision of presynaptic neurotransmitter release (Yoshihara, 2005).

Genetic modifiers of dFMR1 encode RNA granule components in Drosophila

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and this study describes the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP's translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo (Cziko, 2009).

It is suggested, that as for previously identified sev-dfmr1 suppressors Ago1, Lgl, and Me31b, analysis of PABP, Smd3, Rm62, Orb2, and Dco proteins, encoded by the sev-dfmr1 suppressor genes identified in this study, will help elucidate how dFMR1 works in translational regulation, RNA targeting and localization, and ncRNA pathway function (Cziko, 2009).

Three lines of evidence indicate that the genes identified encode proteins with translational repressor activity. First, with the exception of Dco, all of these proteins have been previously implicated in some aspect of RNA metabolism and are present on dFMR1-containing neuritic granules in which RNA is repressed and transported. Second, the rough-eye phenotype observed in sev-dfmr1 has been linked to the ability of FMRP to repress mRNA translation. Thus, it would be expected that the phenotype would be alleviated by mutations that reduce the efficiency of translational repression. Third, overexpression of Dco, Pabp, Orb2, or Rm62 inhibits the dendritic growth of neurons, a phenotype predicted for neuronal translational repressors. These observations are consistent with the idea that translation of RNAs in neurites, which promotes dendritic branching, is inhibited by overexpression of Dco, Pabp, Orb2, or Rm62. Thus, genetic interaction data, molecular localization, and one functional test in dendrites indicate that Dco/Dbt, PABP, Rm62, or SmD3 function as neuronal translational repressors (Cziko, 2009).

The identification of several canonical translational-factor encoding genes as suppressors of sev-dfmr1 highlights the point that individual translational control molecules work in multicomponent complexes and therefore have several functional interactions. PABP is one example of a protein that is currently believed to perform two opposing functions of translational control. In addition to its well-studied role as a translational activator, PABP can mediate translational repression, e.g., of Vasopressin mRNA although the exact mechanism remains unclear. Dual roles in activation and repression are also suggested by the observation that reduced or elevated levels of PABP have similar effects at the Drosophila neuromuscular junction (NMJ). Additionally, PABP associates with particles containing BC1, a neuron-specific noncoding RNA with translational repressor function, as well as a CYFIP-FMRP complex that may function as a repressor in some contexts but as an activator in others. Similarly, Orb2 homologs (CPEBs) though required for translational activation of CPE-containing mRNAs via poly-A polymerase, also allow translational repression in combination with Maskin or Cup proteins (Cziko, 2009).

It was somewhat surprising that SmD3, a splicing factor, was identified in a screen for translational repressors. However, SmD3 has additional nonsplicing functions: in Caenorhabditis elegans, the Sm proteins are required for germ cell mRNP assembly and RNA localization. Such a role in translational regulation and mRNP assembly is more consistent with functions predicted by the genetic experiments (Cziko, 2009).

Rm62/Dmp68 is a member of the DEAD-box helicase family that has been shown to be associated with a dFMR1-containing RNAi silencing complex. It also has additional roles during transcription and mRNA processing as well as potentially in miRNA processing as part of the Drosha complex. Based on the biochemical evidence for Rm62's presence in FMRP-containing complexes, it is not surprising that rm62 mutations show strong genetic interactions with dfmr1. However, the mechanism of suppression remains unknown (Cziko, 2009).

Finally Dco/Dbt, is by far the most elusive protein in regard to its potential function in the translational regulatory pathway. Dco/Dbt, a casein kinase I (CKI) is best known from circadian biology where it phosphorylates Per and expedites its degradation. dFMR1 protein has several phosphorylation sites, one of which in S2 cells has been demonstrated to be phosphorylated by a CKII protein. While the functional requirement for CKI-dependent dFMR1 phosphorylation is as of yet not understood, there is considerable evidence that the phosphorylation state of FMRP may actually determine its role in translation. Biochemical data demonstrate that most FMRP in granules is in the phosphorylated state while FMRP in the polysome fraction is dephosphorylated, suggesting a mechanism to switch state from an activator to a repressor, and an important regulatory role for kinases that phosphorylate FMRP (Cziko, 2009).

Another interesting potential link between the two proteins is the behavioral observation that patients with Fragile-X Mental Retardation often display circadian disturbances. This altered circadian rhythm is also present in the Drosophila dfmr1 mutants that usefully model fragile-X syndrome (Cziko, 2009).

The identification of these proteins as sev-dfmr1 modifiers illustrates the many possibly regulatory roles of RNA-associated proteins. In addition, the data associating Dco/Dbt with RNA regulation indicates unexplored and novel mechanisms of RNA regulation in neurons (Cziko, 2009).

Given that dFMR1/FMRP is thought to function in miRNA-dependent translational repression, it was of particular interest to asking whether these dFMR1 interactors had any role in this pathway. To address this issue, a sensitive in vivo assay that uses a fluorescent reporter was employed to reveal the strength of translational repression via an endogenous (bantam) miRNA. When combined with genetic mosaic analysis, this assay can be used to study null mutations in candidate genes, as long as the mutations do not cause cell lethality. The assay appears more sensitive than typically used cell-based assays on the evidence of prior analysis of Me31B, whose requirement for miRNA function, clearly seen in the in vivo assay, is only evident in double-knockdown experiments in the more commonly used cell-culture assays (Cziko, 2009).

In vivo experiments revealed no requirement for the sev-dfmr1 interacting proteins Dco, Orb2, Rm62, and SmD3 in miRNA repression. For reasons explained above, it is unlikely that this reflects a weakness in the experimental assay for miRNA function. A bigger surprise was the finding that the dFMR1 itself appeared dispensable for miRNA function in vivo. Because the allele used is a well-characterized null allele, and the absence of dFMR1 in the mutant clones is confirmed by antibody staining, the conclusion that dFMR1 is not a core, essential component of the RISC/miRNA pathway is strong. This conclusion is not inconsistent with any of the existing data showing biochemical association between RISC and FMRP and genetic interactions between Ago1 and FMRP. However, it is also consistent with recent observations indicating the dispensability of FMRP for RISC function in cultured cells. It is suggested that the function of dFMR1 and, by extension, FMRP may be restricted to a subset of transcripts, for instance those with UTRs containing both FMRP binding motifs and miRNA target elements. Indeed similar models that account for the mRNA specificity of FMRP have been previously proposed (Cziko, 2009).

These data provide a foundation on which to design further experiments to understand the specific roles of FMR1 and its interacting proteins in translational control (Cziko, 2009).

Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LNvs) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. This study shows that two distinct G protein signaling pathways are required in LNvs for 24 hr rhythms. Reducing signaling in LNvs via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LNvs in culture, it was found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LNvs and that reduced GABA(B)-R3 expression in vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LNv molecular clocks. Therefore, s-LNvs require GABAergic inputs for 24 hr rhythms (Dahdal, 2010).

The long-periods observed with reduced Gs signaling are consistent with four other manipulations of cAMP levels or PKA activity that alter fly circadian behavior. First, long-period rhythms with dnc over-expression complement the short periods of dnc hypomorphs and suggest that the latter are due to loss of dnc from LNvs. dnc mutants also increase phase shifts to light in the early evening. However, this study found no difference in phase delays or advances between Pdf > dnc and control flies, suggesting that altered light-responses of dnc hypomorphs are due to dnc acting in other clock neurons. The period-altering effects seen when manipulating cAMP levels are also consistent with finding stat expressing the cAMP-binding domain of mammalian Epac1 in LNvs lengthens period. This Epac1 domain likely reduces free cAMP levels in LNvs, although presumably not as potently as UAS-dnc. Third, mutations in PKA catalytic or regulatory subunits that affect the whole fly disrupt circadian behavior. Fourth, over-expressing a PKA catalytic subunit in LNvs rescues the period-altering effect of a UAS-shibire transgene that alters vesicle recycling, although the PKA catalytic subunit had no effect by itself. The long periods observed with reduced Gs signaling in LNvs also parallel mammalian studies in which pharmacologically reducing Adenylate cyclase activity lengthened period in SCN explants and mice (Dahdal, 2010).

G-proteins typically transduce extracellular signals. What signals could activate Gs in s-LNvs? PDF is one possibility since PDFR induces cAMP signaling in response to PDF in vitro, indicating that it likely couples to Gs. PDF could signal in an autocrine manner since PDFR is present in LNvs. However, the long-periods observed with reduced Gs signaling differ from the short-period and arrhythmic phenotypes of Pdf and pdfr mutants. The likeliest explanation for these differences is that the altered behavior of Pdf and pdfr mutants results from effects of PDF signaling over the entire circadian circuit, whereas the current manipulations specifically targeted LNvs. Indeed, LNvs are not responsible for the short-period rhythms in Pdf01 null mutant flies. Other possible explanations for the differences between the long-period rhythms with decreased Gs signaling in LNvs and the short-period rhythms of Pdf and pdfr mutants are that additional GPCRs couple to Gs in s-LNvs and influence molecular clock speed and that the current manipulations decrease rather than abolish reception of PDF. In summary, the data shows that Gs signaling via cAMP in s-LNvs modulates period length (Dahdal, 2010).

Go signaling via PLC21C constitutes a novel pathway that regulates the s-LNv molecular clock. This study found that Go and the metabotropic GABAB-R3 receptor are required for the inhibitory effects of GABA on larval LNvs, which develop into adult s-LNvs. The same genetic manipulations that block GABA inhibition of LNvs in culture (expression of Ptx or GABAB-R3-RNAi) lengthened the period of adult locomotor rhythms. Furthermore, the molecular clock in s-LNvs is disrupted when a subset of GABAergic neurons are hyper-excited. Since the LNvs do not produce GABA themselves, s-LNvs require GABAergic inputs to generate 24hr rhythms. Thus s-LNvs are less autonomous for determining period length in DD than previously anticipated (Dahdal, 2010).

Activation of G-proteins can have both short- and long-term effects on a cell. With Go signaling blocked by Ptx, short-term effects on LNv responses were detected in response to excitatory ACh and longer-term effects on the molecular clock. The latter are presumably explained by PLC activation since the behavioral phenotypes of Pdf > GoGTP flies were rescued by reducing Plc21C expression (Dahdal, 2010).

Since s-LNv clocks were unchanged even when the speed of all non-LNv clock neurons were genetically manipulated, it is surprising to find s-LNv clocks altered by signaling from GABAergic non-clock neurons. Why would LNvs need inputs from non-clock neurons to generate 24hr rhythms? One possibility is that LNvs receive multiple inputs which either accelerate or slow down the pace of their molecular clock but overall balance each other to achieve 24hr rhythms in DD. Since reducing signaling by Gs and Go lengthens period, these pathways normally accelerate the molecular clock. According to this model, there are unidentified inputs to LNvs which delay the clock. Identifying additional receptors in LNvs would allow this idea to be tested (Dahdal, 2010).

Previous work showed that GABAergic neurons project to LNvs and that GABAA receptors in l-LNvs regulate sleep. The current data show that constitutive activation of Go signaling dramatically alters behavioral rhythms, suggesting that LNvs normally receive rhythmic GABAergic inputs. But how can s-LNvs integrate temporal information from non clock-containing GABAergic neurons? s-LNvs could respond rhythmically to a constant GABAergic tone by controlling GABAB-R3 activity. Indeed, a recent study found that GABAB-R3 RNA levels in s-LNvs are much higher at ZT12 than at ZT0 (Kula-Eversole, 2010). Strikingly, this rhythm in GABAB-R3 expression is in antiphase to LNv neuronal activity. Thus regulated perception of inhibitory GABAergic inputs could at least partly underlie rhythmic LNv excitability. GABAergic inputs could also help synchronize LNvs as in the cockroach circadian system. Thus GABA's short-term effects on LNv excitability, likely mediated by Gβ/γ, and GABA's longer-term effects on the molecular clock via Go may both contribute to robust rhythms (Dahdal, 2010).

This work adds to the growing network view of circadian rhythms in Drosophila where LNvs integrate information to set period for the rest of the clock network in DD. The period-altering effects of decreased G-protein signaling in LNvs point to a less hierarchical and more distributed network than previously envisioned. Since the data strongly suggests that GABA inputs are novel regulators of 24hr rhythms, the GABAergic neurons that fine-tune the s-LNv clock should be considered part of the circadian network (Dahdal, 2010).

cAMP-dependent protein kinase 1: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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